Amino acids containing dihydropyridine ring systems for site-specific delivery of peptides to the brain

ABSTRACT

The invention provides novel amino acids and peptides containing them which comprise a dihydropyridine -&gt;&lt;- pyridinium salt-type redox system and which provide site-specific and sustained delivery of pharmacologically active peptides to the brain. These new amino acids contain a redox system appended directly or via an alkylene bridge to the carbon atom adjacent to the carboxyl carbon and may be incorporated into a peptide chain at a variety of positions, including non-terminal positions.

FIELD OF THE INVENTION

The invention provides novel amino acids and peptides containing themwhich comprise a dihydropyridine⃡pyridinium salt-type redox system andwhich provide site-specific and sustained delivery of pharmacologicallyactive peptides to the brain.

BACKGROUND OF THE INVENTION

The delivery of drug species to the brain is ofttimes seriously limitedby transport and metabolism factors and, more specifically, by thefunctional barrier of the endothelial brain capillary wall, i.e. theblood-brain barrier or BBB. Site-specific delivery and sustaineddelivery of drugs to the brain are even more difficult.

Indeed, the barriers separating plasma from the brain and cerebrospinalfluid (CSF) are complex systems involving passive and active transportand serve a number of important functions. The boundary between plasmaand the central nervous system (CNS) is much less permeable than thatbetween plasma and other tissue cells to a variety of water solublesubstances, such as organic electrolytes, organic acids and bases, aswell as to large molecules such as proteins. Such a barrier alsoprovides a path for clearance from the brain of the breakdown productsof cellular metabolism. The CNS and its fluids can be consideredbasically a three-compartment system: the blood or the plasma, CSF andbrain tissue. There is a diffusion-controlled exchange between CSF andthe extracellular fluid (CF) of the brain. It has also been suggestedthat the permeabilities of blood-CSF and blood-brain barriers arepractically identical with respect to drugs and other foreignsubstances. Mayer et al, J. Pharmacol. and Exp. Therap., 125, 185(1959).

The BBB is, moreover, basically the result of the fact that theendothelial cells in the brain capillaries are joined by continuous,tight intercellular junctions, such that material has to pass throughthe cells rather than between them in order to move from blood to brain.It is interesting that there are areas within the brain, such as thesubfornical body and the postremia, in which the capillary cells are notclosely linked so that they lack the characteristics of the BBB. Theyprovide the entry of small amounts of compounds which would notordinarily enter the barriers. Hoffman and Olszewzki, Neurology(Minneap.), 11, 1081 (1961).

Foreign compounds which enter organs other than the central nervoussystem with ease, may penetrate the CNS slowly or hardly at all. Anumber of theories concerning the nature of the barrier have beenproposed. The widely accepted concept describes the boundary as afat-like layer interspersed with small pores, although the BBB is not asimple, anatomically well-defined unitary physical entity. ShuttleworthProg. Exp. Tumor Res., 17, 279 (1972). Penetration of such a barrier mayoccur by several processes: lipid soluble substances may passivelypenetrate into the cells, while small molecules such as water and ureamay pass through the pores. In addition to these simple physicalprocesses, carrier-mediated and active transport processes govern themovement of many molecules through the BBB. Thus, it is generallyaccepted that lipid solubility, degree of ionic dissociation orprotonation and the ability of temporary combination with membraneconstituents affect delivery through the BBB. It has been shown, forexample, that in the class of barbiturates, a quantitative correlationcould be established between their ease to pass into the brain (asreflected by the different times of onset of anesthetic action) andtheir lipid/water partition coefficient. Mark et al, J. Pharmacol. andExp. Therap., 123, 79 (1957). The role of lipid solubility in drugpenetration through the BBB is also exemplified by the better absorptionof the sparingly water-soluble thiamine propyl disulfide (TPD) ascompared to the water-soluble thiamine hydrochloride (THCl). Thomson etal, Ann. Int. Med., 74, 529 (1971). Some materials such as glucose andamino acids are transported by active mechanism, characterized bysaturation, bidirectional molecular specificity, bidirectionalcompetitive inhibition and bidirectional countertransport. Fishman, Am.J. Physiol., 206, 836 (1964).

Changes in permeability of the BBB can be caused by several pathologicaland toxicological processes. Pardridge, Connor and Crawford, CRC Crit.Rev. Toxicol., 179 (1975). A general increase in the barrierpermeability, such as a nonspecific breakdown of the barrier has,however, several consequences, including cerebral edema.

It too is well documented that the BBB is relatively impermeable to theionized forms of drugs and other molecules. Drugs which are weak organicelectrolytes appear to pass from blood to CSF to reach a steady stateratio characteristic of each molecule according to its pK_(a) and theexistence of a normal pH gradient between blood and CSF. It is clearthat it is the most difficult for quaternary pyridinium or ammoniumsalts to penetrate the BBB.

And removal of substances from the brain and CSF is obviously asignificant factor in regulating drug concentrations in the CNS. Thereare several efflux processes: bulk flow via the arachnoid villi,diffusion of lipid soluble substances into brain and blood, activetransport and metabolism by adjacent meninges. Once a drug or metaboliteenters the CSF from blood or brain by simple diffusion, it may rapidlybe removed, either by nonselective bulk flow or by active transportmechanism associated with the choroid plexus or other nondefinedstructures in the CSF compartment. It is generally accepted that highlylipid-soluble drugs leave the CSF more rapidly than poorly lipid-solubleones, but the barrier to passage of compounds from CSF has onlysuperficial similarity to the blood-CSF barrier.

Drug elimination processes from the brain are significantly directlyrelated to drug accumulation in the brain. It is generally assumed thatefflux in the opposite direction involves almost the same processes asfor entry, except that the role of the bulk flow and the metabolicprocesses in the brain are not to be overlooked.

The two elimination processes studied in the earlier literature andwhich can be said to have a certain bearing on the present inventioninvolve elimination from the brain of ionic species. Thus, it is foundthat non-metabolized ionic species, such as the acetate ion, have athree times slower elimination rate from the CSF than from the blood.Freundt, Arz. Forsch., 23, 949 (1973). An even more dramatic change inthe elimination rate was found in the case of a quaternary piperidiniumsalt. The quaternary salt, formed in situ after delivery of ahaloalkylamine, which undergoes cyclization to the quaternary salt inthe brain as well, was found to have an at least ten times slowerelimination rate from the brain than from the rest of the body. It isconcluded by the authors [Ross and Froden, Eur. J. Pharmacol., 13, 46(1970)] that the outflow rate of the quaternary salt corresponded to theinflow rate. Similar results were obtained for the erythrocytes: theefflux of the quaternary salt was very slow. Ross, J. Pharm. Pharmacol.,27, 322 (1975).

A dihydropyridine⃡pyridinium redox system has recently been successfullyapplied to delivery to the brain of a number of drugs. Generallyspeaking, according to this system, a dihydropyridine derivative of abiologically active compound is synthesized, which derivative can enterthe CNS through the blood-brain barrier following its systemicadministration. Subsequent oxidation of the dihydropyridine species tothe corresponding pyridinium salt leads to delivery of the drug to thebrain.

Three main approaches have been used thus far for delivering drugs tothe brain using this redox system. The first approach involvesderivation of selected drugs which contain a pyridinium nucleus as anintegral structural component. This approach was first applied todelivering to the brain N-methylpyridinium-2-carbaldoxime chloride(2-PAM), the active nucleus of which constitutes a quaternary pyridiniumsalt, by way of the dihydropyridine latentiated prodrug form thereof.Thus, a hydrophilic compound (2-PAM) was made lipoidal (i.e. lipophilic)by making its dihydropyridine form (Pro-2-PAM) to enable its penetrationthrough lipoidal barriers. This simple prodrug approach allowed thecompound to get into the brain as well as other organs, but thismanipulation did not and could not result in any brain specificity. Onthe contrary, such approach was delimited to relatively small moleculequaternary pyridinium ring-containing drug species and did not providethe overall ideal result of brain-specific, sustained release of thedesired drug, with concomitant rapid elimination from the generalcirculation, enhanced drug efficacy and decreased toxicity. No"trapping" in the brain of the 2-PAM formed in situ resulted, andobviously no brain-specific, sustained delivery occurred as anyconsequence thereof: the 2-PAM was eliminated as fast from the brain asit was from the general circulation and other organs. Compare U.S. Pat.Nos. 3,929,813 and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5,685 (1978). See also Bodor, "Novel Approaches for the Design of MembraneTransport Properties of Drugs", in Design of BiopharmaceuticalProperties Through Prodrugs and Analogs, Roche, E. B. (ed.), APhAAcademy of Pharmaceutical Sciences, Washington, D.C., 98-135 (1976).Subsequent extension of this first approach to delivering a much largerquaternary salt, berberine, to the brain via its dihydropyridine prodrugform was, however, found to provide sitespecific sustained delivery tothe brain of that anticancer agent. See Bodor et al, Science, Vol. 214,Dec. 18, 1981, pp. 1370-1372.

The second approach for delivering drugs to the brain using the redoxsystem involves the use of a dihydropyridine/pyridinium carrierchemically linked to a biologically active compound. Bodor et al,Science, Vol. 214, Dec. 18, 1981, pp. 1370-1372, outlines a scheme forthis specific and sustained delivery of drug species to the brain, asdepicted in the following Scheme 1: ##STR1## According to the scheme inScience, a drug [D] is coupled to a quaternary carrier [QC]⁺ and the[D-QC]⁺ which results is then reduced chemically to the lipoidal dihydroform [D-DHC]. After administration of [D-DHC] in vivo, it is rapidlydistributed throughout the body, including the brain. The dihydro form[D-DHC] is then in situ oxidized (rate constant, k₁) (by the NAD⃡NADHsystem) to the ideally inactive original [D-QC]⁺ quaternary salt which,because of its ionic, hydrophilic character, should be rapidlyeliminated from the general circulation of the body, while theblood-brain barrier should prevent its elimination from the brain (K₃>>k₂ ; k₃ >>k₇). Enzymatic cleavage of the [D-QC]⁺ that is "locked" inthe brain effects a sustained delivery of the drug species [D], followedby its normal elimination (k₅), metabolism. A properly selected carrier[QC]⁺ will also be rapidly eliminated from the brain (k.sub. 6 >>k₂).Because of the facile elimination of [D-QC]⁺ from the generalcirculation, only minor amounts of drug are released in the body (k₃>>k₄); [D] will be released primarily in the brain (k₄ >k₂). The overallresult ideally will be a brain-specific sustained release of the targetdrug species. Specifically, Bodor et al worked with phenylethylamine asthe drug model. That compound was coupled to nicotinic acid, thenquaternized to give compounds of the formula ##STR2## which weresubsequently reduced by sodium dithionite to the corresponding compoundsof the formula ##STR3## Testing of the N-methyl derivative in vivosupported the criteria set forth in Scheme 1. Bodor et al speculatedthat various types of drugs might possibly be delivered using thedepicted or analogous carrier systems and indicated that use ofN-methylnicotinic acid esters and amides and their pyridinering-substituted derivatives was being studied for delivery of amino- orhydroxyl-containing drugs, including small peptides, to the brain. Noother possible specific carriers were disclosed. Other reports of thiswork with the redox carrier system have appeared in The Friday EveningPost, Aug. 14, 1981, Health Center Communications, University ofFlorida, Gainesville, Florida; Chemical & Engineering News, Dec. 21,1981, pp. 24-25; and Science News, Jan. 2, 1982, Vol. 121, No. 1, page7. More recently, the redox carrier system has been substantiallyextended in terms of possible carriers and drugs to be delivered. SeeInternational patent application No. PCT/US83/00725, filed May 12, 1983and published Nov. 24, 1983 under International Publication No.W083/03968. Also see Bodor et al, Pharmacology and Therapeutics, Vol.19, No. 3, pp. 337-386 (1983); and Bodor U.S. Pat. No. 4,540,564, issuedSept. 10, 1985.

The aforementioned Bodor U.S. Pat. No. 4,540,564 specificallycontemplates application of the dihydropyridine⃡pyridinium salt carriersystem to amino acids and peptides, particularly small peptides having 2to 20 amino acid units. Among the amino acids and peptides mentioned inthe patent are GABA, tyrosine, tryptophan, met⁵ -enkephalin, leu⁵-enkephalin, LHRH and its analogs and others. Representativecarrier-linked amino acids and peptides illustrated in the Bodor patentare the following:

      AMINO ACID/PEPTIDE CARRIER-DRUG (QUATERNARY) CARRIER-DRUG (DIHYDRO)      ##STR4##      ##STR5##      ##STR6##       NH.sub.2CH.sub.2 CH.sub.2 CH.sub.2      COOH(GABA)     ##STR7##      ##STR8##       TyrGlyGlyPheLeu(leu.sup.5      -enkephalin)     ##STR9##      (not depicted)

Thus, in the depicted carrier system as applied to amino acids andpeptides, the free carboxyl function is suitably protected to preventpremature metabolism while the trigonelline-type carrier is linked tothe amino acid or peptide through its free amino function. Oxidation ofthe dihydropyridine carrier moiety in vivo to the ionic pyridinium saltcarrier/drug entity prevents elimination thereof from the brain, whileelimination from the general circulation is accelerated, and subsequentcleavage of the quaternary carrier/drug species results in sustaineddelivery of the amino acid or peptide (e.g. tryptophan, GABA, leu⁵-enkephalin, etc.) in the brain and facile elimination of the carriermoiety.

The third approach for delivering drugs to the brain using the redoxsystem provides derivatives of centrally acting amines in which aprimary, secondary or tertiary amine function has been replaced with adihydropyridine/pyridinium salt redox system. These brain-specificanalogs of centrally acting amines have been recently described inInternational patent application No. PCT/US85/00236, filed Feb. 15, 1985and published Sept. 12, 1985 under International Publication No.W085/03937. The dihydropyridine analogs are characterized by thestructural formula ##STR10## wherein D is the residue of a centrallyacting primary, secondary or tertiary amine, and ##STR11## is a radicalof the formula ##STR12## wherein the dotted line in formula (a)indicates the presence of a double bond in either the 4 or 5 position ofthe dihydropyridine ring; the dotted line in formula (b) indicates thepresence of a double bond in either the 2 or 3 position of thedihydroquinoline ring system; m is zero or one; n is zero, one or two; pis zero, one or two, provided that when p is one or two, each R informula (b) can be located on either of the two fused rings; q is zero,one, or two, provided that when q is one or two, each R in formula (c)can be located on either of the two fused rings; and each R isindependently selected from the group consisting of halo, C₁ -C₇ alkyl,C₁ -C₇ alkoxy, C₂ -C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁ -C₇haloalkyl, C₁ -C₇ alkylthio, C₁ -C₇ alkylsulfinyl, C₁ -C₇ alkylsulfonyl,--CH═NOR"' wherein R"' is H or C₁ -C₇ alkyl, and --CONR'R" wherein R'and R", which can be the same or different, are each H or C₁ -C₇ alkyl.These dihydropyridine analogs act as a delivery system for thecorresponding biologically active quaternary compounds in vivo. Due toits lipophilic nature, the dihydropyridine analog will distributethroughout the body and has easy access to the brain through theblood-brain barrier. Oxidation in vivo will then provide the quaternaryform, which will be "locked" preferentially in the brain. Incontradistinction to the drug-carrier entities described in Bodor U.S.Pat. No. 4,540,564 and related publications, however, there is noreadily metabolically cleavable bond between drug and quaternaryportions, and the active species delivered is not the original drug fromwhich the dihydro analog was derived, but rather is the quaternaryanalog itself.

The aforementioned International Publication No. W085/03937 contemplatesapplication of its analog system to amino acids and small peptides, e.g.the enkephalins, tryptophan, GABA, LHRH analogs and others. Illustratedredox analogs include the following:

    __________________________________________________________________________    AMINO ACID/PEPTIDE   QUATERNARY ANALOG    DIHYDROPYRIDINE                     __________________________________________________________________________                                              ANALOG                               ##STR13##                                                                                                             ##STR14##                             ##STR15##                                                                    NH.sub.2CH.sub.2 CH.sub.2 CH.sub.2 COOH (GABA)                                                      ##STR16##                                                                                          ##STR17##                           ##STR18##                                                                                          ##STR19##                                                                                          ##STR20##                           ##STR21##                                                                                          ##STR22##                                                                                          ##STR23##                          __________________________________________________________________________

In the depicted analog system as applied to amino acids and peptides,the free carboxyl function is thus suitably protected to preventpremature metabolism while the dihydropyridine⃡pyridinium salt type redoxsystem replaces the free amino function in the amino acid or peptide.

As described in International Publication No. W085/03937, the chemicalprocesses for preparing the redox analog derivatives replace any freeamino function in the selected drug with the redox analog system. Whenthese processes are applied to amino acids, they provide a redox aminoacid which no longer contains a free amino function for linkage toanother amino acid or peptide via a peptide bond (--CONH--). Such ananalog amino acid can thus only be used to prepare a peptide having theanalog amino acid located at the peptide's N-terminus. This severelylimits use of the redox analog amino acids in peptide synthesis. Ittherefore would be desirable to provide a new approach for deliveringpeptides to the brain using the redox system, which approach wouldprovide novel redox amino acids which could be used to synthesizepeptides having the redox system inserted at a variety of locations inthe peptide chain.

SUMMARY AND OBJECTS OF THE INVENTION

Accordingly, a major object of the present invention is the provision ofa new approach for delivering peptides to the brain using the redoxsystem. This approach provides novel amino acids and peptides containingthem which comprise a dihydropyridine⃡pyridinium salt redox system. Thesenew amino acids contain the redox system appended directly or via analkylene bridge to the carbon atom adjacent to the carboxyl carbon. Theredox amino acids of this invention are useful in the preparation ofnovel, biologically active peptides which are brain-specific and whichprovide sustained in-brain activity, including many peptides in whichthe redox amino acid is located in a non-terminal position of thepeptide chain.

Consistent with the foregoing, the present invention provides novelamino acids which, in the reduced form, have the structural formula##STR24## wherein Z is either a direct bond or C₁ -C₆ alkylene and canbe attached to the heterocyclic ring via a ring carbon atom or via thering nitrogen atom; R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₂aralkyl when Z is attached to a ring carbon atom; R₁ is a direct bondwhen Z is attached to the ring nitrogen atom; R₂ and R₃, which can bethe same or different, are selected from the group consisting ofhydrogen, halo, cyano, C₁ -C₇ alkyl, C₁ -C₇ alkoxy, C₂ 14 C₈alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁ -C₇ haloalkyl, C₁ -C₇ alkylthio,C₁ -C₇ alkylsulfinyl, C₁ -C₇ alkylsulfonyl, --CH═NOR'" wherein R'" ishydrogen or C₁ -C₇ alkyl, and --CONR'R" wherein R' and R", which can bethe same or different, are each hydrogen or C₁ -C₇ alkyl; or one of R₂and R₃ together with the adjacent ring carbon atom forms a benzene ringfused to the heterocyclic ring, which benzene ring may optionally bearone or two substituents, which can be the same or different, selectedfrom the group consisting of hydroxy, protected hydroxy, halo, cyano, C₁-C₇ alkyl, C₁ -C₇ alkoxy, C₂ -C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁-C₇ haloalkyl, C₁ -C₇ alkylthio, C₁ -C₇ alkylsulfinyl, C₁ -C₇alkylsulfonyl, --CH═NOR'" wherein R'" is hydrogen or C₁ -C₇ alkyl, and--CONR'R" wherein R' and R", which can be the same or different, areeach hydrogen or C₁ -C₇ alkyl; R₄ is hydrogen or carboxyl protectivegroup; R₅ is hydrogen or an amino protective group; and the dotted linesindicate that the compound of formula (I) contains a 1,4- or1,6-dihydropyridine, a 1,4- or 1,2-dihydroquinoline, or a1,2-dihydroisoquinoline ring system.

The non-toxic pharmaceutically acceptable salts of the compounds offormula (I) are also within the ambit of this invention.

The corresponding oxidized or quaternary form of the redox amino acidsof the present invention can be represented by the formula ##STR25##wherein X⁻ is the anion of a non-toxic pharmaceutically acceptable acidand Z, R₁, R₂, R₃, R₄, and R₅ are as defined with formula (I).

The new dihydropyridine and pyridinium salt-type amino acids of formulas(I) and (II) are useful in the preparation of novel redox peptides ofthe partial formulas: ##STR26## wherein Z, R₁, R₂, R₃, X⁻ and the dottedlines are as defined with formulas (I) and (II), the remainder of thepeptide structure being defined hereinbelow.

The new peptide analogs of partial structure (A) act as a deliverysystem for the corresponding quaternary salts of partial structure (B)in vivo; the quaternary derivatives, which also are chemicalintermediates to the dihydro compounds, are pharmacologically active orconvertible in vivo to pharmacologically active peptides, and arecharacterized by site-specific and sustained delivery to the brain whenadministered via the corresponding dihydropyridine form.

DETAILED DESCRIPTION OF THE INVENTION

More particularly in accord with the present invention, the followingdefinitions are applicable:

The term "lipoidal" as used herein is intended to mean lipid-soluble orlipophilic.

The term "C₁ -C₆ alkylene" as used herein encompasses bivalent radicalsof the type --(CH₂)_(n) -- wherein n is 1 to 6, as well as thecorresponding branched chain groups, e.g. methylene, ethylene,propylene, trimethylene, 1,2-butylene, 2,3-butylene, tetramethylene andthe like. Preferably, C₁ -C₆ alkylene is --(CH₂)_(n) -- wherein n is 1or 4.

The term "halo" encompasses fluoro, chloro, bromo and iodo.

The term "C₁ -C₇ alkyl" includes straight and branched lower alkylradicals having up to seven carbon atoms. When R₂ and/or R₃ are C₁ -C₇alkyl, they are preferably methyl or ethyl. When R₁ is C₁ -C₇ alkyl, itis preferably methyl.

The term "C₁ -C₇ alkoxy" includes straight and branched chain loweralkoxy radicals having up to seven carbon atoms. When R₂ and/or R₃ areC₁ -C₇ alkoxy, they are preferably methoxy or ethoxy.

The term "C₂ -C₈ alkoxycarbonyl" designates straight and branched chainradicals of the formula ##STR27## wherein the C₁ -C₇ alkyl group isdefined as above. When R₂ and/or R₃ are alkoxycarbonyl, they arepreferably ethoxycarbonyl or isopropoxycarbonyl.

The term "C₂ -C₈ alkanoyloxy" designates straight and branched chainradicals of the formula ##STR28## wherein the C₁ -C₇ alkyl group isdefined as above. When R₂ and/or R₃ are alkanoyloxy, they are preferablyacetoxy, pivalyloxy or isobutyryloxy.

The term "C₁ -C₇ haloalkyl" designates straight and branched chain loweralkyl radicals having up to seven carbon atoms and bearing one to threehalo substituents (F, Cl, Br or I), which can be the same or different.Specific examples of the contemplated monohaloalkyl and polyhaloalkylgroups include chloromethyl, dichloromethyl, trichloromethyl,bromomethyl, fluoromethyl, difluoromethyl, trifluoromethyl,1-fluoroethyl, 1-chloroethyl, 2-chloroethyl, 2,2,2-trichloroethyl,2,2,2-trifluoroethyl, 1,2-dichloroethyl, 1-chloropropyl, 3-chloropropyl,1-chlorobutyl, 1-chloropentyl, 1-chlorohexyl, 4-chlorobutyl and thelike. Preferably, the haloalkyl group contains 1 or 2 carbon atoms andbears 1 to 3 halogen substituents, e.g. chloromethyl or trifluoromethyl.

The term "C₁ -C₇ alkylthio" includes straight and branched chainradicals of the type

    (C.sub.1 -C.sub.7 alkyl)--S--

wherein C₁ -C₇ alkyl is defined as before. When R₂ and/or R₃ arealkylthio, they are preferably methylthio.

The terms "C₁ -C₇ alkylsulfinyl" and "C₁ -C₇ alkylsulfonyl" designateradicals of the formulas

    (C.sub.1 -C.sub.7 alkyl)--SO--

and

    (C.sub.1 -C.sub.7 alkyl)--SO.sub.2 --,

respectively, wherein C₁ -C₇ alkyl is defined as before. When R₂ and/orR₃ are alkylsulfinyl or alkylsulfonyl, methylsulfinyl and methylsulfonylare preferred.

When R₂ and/or R₃ are --CH═NOR'", they are preferably --CH═NOH or--CH═NOCH₃.

When R₂ and/or R₃ are --CONR'R", they are preferably --CONH₂ or--CON(CH₃)₂.

The term "C₇ -C₁₂ aralkyl" as used herein designates radicals of thetype

    --alkylene--aryl

wherein the aryl portion is phenyl or naphthyl and the alkylene portion,which can be straight or branched, can contain up to 6 carbon atoms,e.g. methylene, ethylene, propylene, trimethylene, 1,2-butylene,2,3-butylene, tetramethylene and the like. When R₁ is aralkyl, it ispreferably benzyl.

The expression "non-toxic pharmaceutically acceptable salts" as usedherein generally includes the non-toxic salts of compounds of formulas(I) and (A) formed with non-toxic, pharmaceutically acceptable inorganicor organic acids HX. For example, the salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glucolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric,methanesulfonic, toluenesulfonic and the like. The expression "anion ofa non-toxic pharmaceutically acceptable acid" as used herein, e.g. inconnection with structures (II) and (B), is intended to include anionsof such organic or inorganic acids HX.

The expression "hydroxyl protective group" as used herein is intended todesignate a group (Y) which is inserted in place of a hydrogen atom ofan OH group or groups in order to protect the OH group(s) duringsynthesis and/or to improve lipoidal characteristics and preventpremature metabolism of the OH group(s) prior to the compound's reachingthe desired site in the body. The expression "protected hydroxysubstituent" designates an OY group wherein Y is a "hydroxyl protectivegroup" as defined above.

Typical hydroxyl protective groups contemplated by the present inventionare acyl groups and carbonates. When the hydroxyl protective group isacyl (i.e., when it is an organic radical derived from carboxylic acidby removal of the hydroxyl group), it preferably represents an acylradical selected from the group consisting of alkanoyl having 2 to 8carbon atoms; alkenoyl having one or two double bonds and 3 to 8 carbonatoms; ##STR29## wherein the cycloalkyl portion contains 3 to 7 ringatoms and r is zero, one, two or three; phenoxyacetyl; pyridinecarbonyl;and ##STR30## wherein r is zero, one, two or three and phenyl isunsubstituted or is substituted by 1 to 3 alkyl each having 1 to 4carbon atoms, alkoxy having 1 to 4 carbon atoms, halo, trifluoromethyl,dialkylamino having 2 to 8 carbon atoms or alkanoylamino having 2 to 6carbon atoms.

When the acyl group is alkanoyl, there are included both unbranched andbranched alkanoyl, for example, acetyl, propionyl, butyryl, isobutyryl,valeryl, isovaleryl, 2-methylbutanoyl, pivalyl (pivaloyl),3-methylpentanoyl, 3,3-dimethylbutanoyl, 2,2-dimethylpentanoyl and thelike. Pivalyl, isobutyryl and isovaleryl are especially preferred.

When the acyl group is alkenoyl, there are included, for example,crotonyl, 2,5-hexadienoyl and 3,6-octadienoyl.

When the acyl group is ##STR31## there are included cycloalkanecarbonyland cycloalkanealkanoyl groups wherein the cycloalkane portion canoptionally bear 1 or 2 alkyl groups as substituents, e.g.cyclopropanecarbonyl, 1-methylcyclopropanecarbonyl, cyclopropaneacetyl,α-methylcyclopropaneacetyl, 1-methylcyclopropaneacetyl,cyclopropanepropionyl, α-methylcyclopropanepropionyl,2-isobutylcyclopropanepropionyl, cyclobutanecarbonyl,3,3-dimethylcyclobutanecarbonyl, cyclobutaneacetyl,2,2-dimethyl-3-ethylcyclobutaneacetyl, cyclopentanecarbonyl,cyclohexaneacetyl, cyclohexanecarbonyl, cycloheptanecarbonyl andcycloheptanepropionyl. Cyclohexanecarbonyl is especially preferred.

When the acyl group is pyridinecarbonyl, there are included picolinoyl(2-pyridinecarbonyl), nicotinoyl (3-pyridinecarbonyl) and isonicotinoyl(4-pyridinecarbonyl).

When the acyl group is ##STR32## there are included, for example,benzoyl, phenylacetyl, α-phenylpropionyl, β-phenylpropionyl, p-toluyl,m-toluyl, o-toluyl, o-ethylbenzoyl, p-tert-butylbenzoyl,3,4-dimethylbenzoyl, 2-methyl-4-ethylbenzoyl, 2,4,6-trimethylbenzoyl,m-methylphenylacetyl, p-isobutylphenylacetyl,β-(p-ethylphenyl)propionyl, p-anisoyl, m-anisoyl, o-anisoyl,m-isopropoxybenzoyl, p-methoxyphenylacetyl, m-isobutoxyphenylacetyl,m-diethylaminobenzoyl, 3-methoxy-4-ethoxybenzoyl,3,4,5-trimethoxybenzoyl, p-dibutylaminobenzoyl,3,4-diethoxyphenylacetyl, β-(3,4,5-trimethoxyphenyl)propionyl,o-iodobenzoyl, m-bromobenzoyl, p-chlorobenzoyl, p-fluorobenzoyl,2-bromo-4-chlorobenzoyl, 2,4,6-trichlorobenzoyl, p-chlorophenylacetyl,α-(m-bromophenyl)propionyl, p-trifluoromethylbenzoyl,2,4-di(trifluoromethyl)benzoyl, m-trifluoromethylphenylacetyl,β-(3-methyl-4-chlorophenyl)propionyl, p-dimethylaminobenzoyl,p-(N-methyl-N-ethylamino)benzoyl, o-acetamidobenzoyl,m-propionamidobenzoyl, 3-chloro-4-acetamidophenylacetyl,p-n-butoxybenzoyl, 2,4,6-triethoxybenzoyl,β-(p-trifluoromethylphenyl)propionyl, 2-methyl-4-methoxybenzoyl,p-acetamidophenylpropionyl, and 3-chloro-4-ethoxybenzoyl.

When the hydroxyl protective group is a carbonate grouping, it has thestructural formula ##STR33## i.e., it is an organic radical which can beconsidered to be derived from a carbonic acid by removal of the hydroxylgroup from the COOH portion. Y' preferably represents alkyl having 1 to7 carbon atoms; alkenyl having one or two double bonds and 2 to 7 carbonatoms;

    cycloalkyl--C.sub.r H.sub.2r --

wherein the cycloalkyl portion contains 3 to 7 ring atoms and r is zero,one, two or three; phenoxy; 2-, 3-, or 4-pyridyl; or

    phenyl--C.sub.r H.sub.2r --

wherein r is zero, one, two or three and phenyl is unsubstituted or issubstituted by 1 to 3 alkyl each having 1 to 4 carbon atoms, alkoxyhaving 1 to 4 carbon atoms, halo, trifluoromethyl, dialkylamino having 2to 8 carbon atoms or alkanoylamino having 2 to 6 carbon atoms. Mostpreferably, Y' is C₁ -C₇ alkyl, particularly ethyl or isopropyl.

Similarly, the expression "carboxyl protective group" as used herein isintended to designate a group (W) which is inserted in place of ahydrogen atom of a COOH group or groups in order to protect the COOHgroup(s) during synthesis and/or to improve lipoidal characteristics andprevent premature metabolism of said COOH group or groups prior to thecompound's reaching the desired site in the body. Typical of suchcarboxyl protective groups W are the groups encompassed by Y' above,especially C₁ -C₇ alkyl, particularly ethyl, isopropyl and t-butyl.While such simple alkyl esters and the like are often useful, othercarboxyl protecting groups may be selected, e.g. in order to achievegreater control over the rate of in vivo hydrolysis of the ester back tothe acid and thus enhance drug delivery. To that end, carboxylprotecting groups W such as the following may be used to replace thehydrogen of the --COOH group: ##STR34## wherein alk is C₁ -C₆ straightor branched alkylene and the alkyl radical is straight or branched andcontains 1 to 7 carbon atoms (e.g. ##STR35##

Other carboxyl protective groups W which can be used to replace thehydrogen of the --COOH group and which are especially useful herein arethe following:

    C.sub.3 -C.sub.12 cycloalkyl--C.sub.p H.sub.2p --

wherein p is 0, 1, 2 or 3;

    C.sub.6 -C.sub.28 polycycloalkyl--C.sub.p H.sub.2p --

wherein p is defined as above;

    C.sub.6 -C.sub.28 polycycloalkenyl--C.sub.p H.sub.2p --

wherein p is defined as above;

    C.sub.3 -C.sub.12 cycloalkenyl--C.sub.p H.sub.2p --

wherein p is defined as above;

    --CH.sub.2 --X.sub.a --R.sub.a

wherein X_(a) is S, SO or SO₂ and R_(a) is C₁ -C₇ alkyl or C₃ -C₁₂cycloalkyl; ##STR36## wherein R_(a) is defined as above; ##STR37##wherein X_(a) is defined as above, R_(b) is C₁ -C₇ alkyl and R_(c) is C₁-C₇ alkyl or wherein R_(b) and R_(c) taken together represent--(CH₂)_(m') -- wherein m' is 3 or 4 and --(CH₂)_(m') -- is optionallysubstituted by one to three C₁ -C₇ alkyl; ##STR38## wherein R_(d) ishydrogen or C₁ -C₇ alkyl and R_(e) is unsubstituted or substituted C₁-C₁₂ alkyl [e.g. ##STR39## C₃ -C₁₂ cycloalkyl--C_(p) J_(2p) -- wherein pis defined as above, C₃ -C₁₂ cycloalkenyl--C_(p) H_(2p) -- wherein p isdefined as above or C₂ -C₈ alkenyl, the substituents being selected fromthe group consisting of halo, C₁ -C₇ alkoxy, C₁ -C₇ alkylthio, C₁ -C₇alkylsulfinyl, C₁ -C₇ alkylsulfonyl, ##STR40## or R_(e) is unsubstitutedor substituted phenyl or benzyl, the substituents being selected fromthe group consisting of C₁ -C₇ alkyl, C₁ -C₇ alkoxy, halo, carbamoyl, C₂-C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy. C₁ -C₇ haloalkyl, mono(C₁ -C₇alkyl)amino, di(C₁ -C₇ alkyl)amino, mono(C₁ -C₇ alkyl)carbamoyl, di(C₁-C₇ alkyl)carbamoyl, C₁ -C₇ alkylthio, C₁ -C₇ alkylsulfinyl and C₁ -C₇alkylsulfonyl, or R_(e) is C₆ -C₂₈ polycycloalkyl--C_(p) H_(2p) -- or C₆-C₂₈ polycycloalkenyl--C_(p) H_(2p) -- wherein p is defined as above;##STR41## wherein R_(d) and R_(e) are defined as above; and ##STR42##wherein R_(d) is defined as above and R_(f) and R_(g), which can be thesame or different, are each hydrogen, C₁ -C₇ alkyl, C₃ -C₁₂cycloalkyl--C_(p) H_(2p) --, C₃ -C₁₂ cycloalkenyl--C_(p) H_(2p) --,phenyl or benzyl, or one of R_(f) and R_(g) is hydrogen, C₁ -C₇ alkyl,C₃ -C₁₂ cycloalkyl--C_(p) H_(2p) --, C₃ -C₁₂ cycloalkenyl--C_(p) H_(2p)--, phenyl or benzyl and the other of R_(f) and R_(g) is C₆ -C₂₈polycycloalkyl--C_(p) H_(2p) -- or C₆ -C₂₈ polycycloalkenyl--C_(p)H_(2p) --, or R_(f) and R_(g) are combined such that --NR_(f) R_(g)represents the residue of a saturated monocyclic secondary amine.

When the carboxyl protecting group is C₃ -C₁₂ cycloalkyl--C_(p) H_(2p)-- or otherwise contains a C₃ -C₁₂ cycloalkyl group, the cycloalkylgroups contain 3 to 8 ring atoms and may optionally bear one or more,preferably one to four, alkyl substituents. Exemplary such cycloalkylgroups are cyclopropyl, 2-methylcyclopropyl, 3-ethylcyclopropyl,2-butylcyclopropyl, 3-pentylcyclopropyl, 2-hexylcyclopropyl, cyclobutyl,2-methylcyclobutyl, 2,3-dimethylcyclobutyl, 3-butylcyclobutyl,4-hexylcyclobutyl, 2,3,3-trimethylcyclobutyl,3,3,4,4-tetramethylcyclobutyl, cyclopentyl, 2-methylcyclopentyl,3-ethylcyclopentyl, 4-butylcyclopentyl, 5-methylcyclopentyl,3-pentylcyclopentyl, 4-hexylcyclopentyl, 2,3-dimethylcyclopentyl,2,2,5,5-tetramethylcyclopentyl, 2,3,4-trimethylcyclopentyl,2,4-dimethyl-3-ethylcyclopentyl, 2,2,3,4,4-pentamethylcyclopentyl,2,3-dimethyl-3-propylcyclopentyl, cyclohexyl, 2,6-dimethylcyclohexyl,3,3,5,5-tetramethylcyclohexyl, 2-methylcyclohexyl, 2-ethylcyclohexyl,4-propylcyclohexyl, 5-butylcyclohexyl, 2,3-dimethylcyclohexyl,2,4-dimethylcyclohexyl, 2,5-dimethylcyclohexyl,2,3,4-trimethylcyclohexyl, 2,3-dimethyl-5-ethylcyclohexyl,2,5-dimethyl-6-propylcyclohexyl, 2,4-dimethyl-3-butylcyclohexyl,2,2,4,4-tetramethylcyclohexyl, 3,3,6,6-tetramethylcyclohexyl,3,3,4,5,5-pentamethylcyclohexyl, 3,3,4,5,5,6-hexamethylcyclohexyl,3,3,5-trimethyl- 4-ethylcyclohexyl, 3,4,4-trimethyl-5-propylcyclohexyl,cycloheptyl, 3-methylcycloheptyl, 5-propylcycloheptyl,6-butylcycloheptyl, 7-methylcycloheptyl, cyclooctyl, 2-methylcyclooctyl,3-ethylcyclooctyl, 3,3,4-trimethylcyclooctyl,3,3,5,5-tetramethylcyclooctyl and the like. Among the presentlypreferred cycloalkyl--C_(p) H_(2p) -- carboxyl protecting groups arecyclohexyl, 2,6-dimethylcyclohexyl and 3,3,5,5-tetramethylcyclohexyl.Thus, when the carboxyl protective group is cycloalkyl--C_(p) H_(2p) --,p is preferably zero or one, most preferably zero.

When the carboxyl protecting group is C₃ -C₁₂ cycloalkenyl--C_(p) H_(2p)-- or otherwise contains a C₃ -C₁₂ cycloalkenyl group, the correspondingunsaturated radicals such as cyclopentenyl and cyclohexenyl and the likeare contemplated. Again, when the protective group is C₃ -C₁₂cycloalkenyl--C_(p) H_(2p) --, p is preferably zero or one, mostpreferably zero.

The polycycloalkyl--C_(p) H_(2p) -- radicals which can serve as carboxylprotective groups, or as portions of carboxyl protective groups, arebridged or fused saturated alicyclic hydrocarbon systems consisting oftwo or more rings, optionally bearing one or more alkyl substituents andhaving a total of 6 to 28 carbon atoms in the ring portion. Thecorresponding bridged or fused unsaturated alicyclic hydrocarbon systemsare intended by the term "C₆ -C₂₈ polycycloalkenyl--C_(p) H_(2p) --".Such polycycloalkyl and polycycloalkenyl radicals are exemplified byadamantyl (especially 1- or 2-adamantyl), adamantylmethyl (especially1-adamantylmethyl), adamantylethyl (especially 1-adamantylethyl),bornyl, norbonyl, (e.g. exo-norbonyl or endo-norbornyl), norbornenyl(e.g. 5-norbornen-2-yl), norbornylmethyl (e.g. 2-norbornylmethyl) andnorbornylethyl (e.g. 2-norbornylethyl), and by radicals of the type

    --C.sub.p H.sub.2p --(sterol residue)

wherein p is defined as above and the sterol residue is the portion of asteroidal alcohol which remains after removal of a hydrogen atom from ahydroxy group therein. The sterol residue is preferably that of apharmacologically inactive steroid, e.g. cholesterol, a bile acid(cholic acid or related compound) or the like. In the case of polycyclicradicals, p is preferably 0, 1 or 2.

When the carboxyl protective group is ##STR43## wherein --NR_(f) R_(g)represents the residue of a saturated monocyclic secondary amine, suchmonocycles preferably have 5 to 7 ring atoms optionally containinganother hetero atom (--O--, --S-- or --N--) in addition to the indicatednitrogen atom, and optionally bear one or more substituents such asphenyl, benzyl and methyl. Illustrative of residues of saturatedmonocyclic secondary amines which are encompassed by the --NR_(f) R_(g)term are morpholino, 1-pyrrolidinyl, 4-benzyl-1-piperazinyl,perhydro-1,2,4-oxathiazin-4-yl, 1- or 4-piperazinyl,4-methyl-1-piperazinyl, piperidino, hexamethyleneimino,4-phenylpiperidino, 2-methyl-1-pyrazolidinyl, 1- or 2-pyrazolidinyl,3-methyl-1-imidazolidinyl, 1- or 3-imidazolidinyl, 4-benzylpiperidinoand 4-phenyl-1-piperazinyl.

Carboxyl protecting groups of the type --C₂ COOR in which R is linear orbranched C₁ -C₁₂ alkyl or C₅ -C₁₆ mono-alicyclic or polycyclic aredescribed in Laruelle et al U.S. Pat. No. 4,568,754. Those groups aresaid to be useful in providing derivatives of 5-hydroxytryptophan(5-HTP) which are resistant to peripheral decarboxylation and whichregenerate 5-HTP at the cerebral level, in this way increasing passagethrough the blood-brain barrier. The Laruelle et al derivatives lack theinstant dihydropyridine⃡pyridinium salt redox systems and thus would notbe capable of providing sustained brain delivery via a "locked-in"quaternary form.

As yet another alternative in accord with the present invention, thecarboxyl group can be protected by converting it to an amide, i.e. the--COOH group is converted to a --CONR_(f) R_(g) group wherein R_(f) andR_(g) are as defined and exemplified above. Such amide groups are alsointended to be encompassed by the expression "carboxyl protecting group"as used with formulas (I) and (II) herein, or similar expressions usedherein in conjunction with peptides of partial structures (A) and (B).

Selection of an appropriate carboxyl protecting group will depend uponthe reasons for protection and the ultimate use of the protectedproduct. For example, if the protecting group is intended to be presentin a pharmaceutically useful peptide end product, it will be selectedfrom those protecting groups described hereinabove which offer lowtoxicity and the desired degree of lipophilicity and rate of in vivocleavage. On the other hand, if the protecting group is used solely forprotection during synthesis, then only the usual synthetic requirementswill generally apply.

Carboxyl protecting groups for use in peptide synthesis are well-knownto those skilled in the art. See, for example, M. Bodanszky, Principlesof Peptide Synthesis, Springer-Verlag, New York 1984, Ives U.S. Pat. No.4,619,915 and the various publications on peptide chemistry referred toin the Ives patent. See also Methoden der Organischen Chemie,Houben-Weyl, Volume 15/1 for protecting groups and Volume 15/2 formethods of peptide synthesis. Representative carboxyl protecting groupsfor synthetic purposes include various silyl esters (e.g. trialkylsilyland trihalosilyl esters), alkyl esters (e.g. tert-butyl esters), benzylesters and the other carboxyl protecting groups mentioned in the Ivespatent. Simple alkyl esters are frequently satisfactory for syntheticpurposes, while bulkier and more complex ester groupings (e.g. those ofthe polycycloalkyl and polycycloalkenyl, such as the sterol, residuetypes) or amides are generally preferred for use in vivo.

The expression "amino protective group" as used herein is intended todesignate a group (T) which is inserted in place of a hydrogen atom ofan amino group or groups in order to protect the amino group(s) duringsynthesis and/or to improve lipoidal characteristics and preventpremature metabolism of said amino group or groups prior to thecompound's reaching the desired site in the body.

As with the carboxyl protecting groups, selection of a suitable aminoprotecting group will depend upon the reason for protection and theultimate use of the protected product. When the protecting group is usedsolely for protection during synthesis, then a conventional aminoprotecting group may be employed. Appropriate amino protecting groupsare known in the art and are described, for example, in the Bodanszky,Ives and Houben-Weyl references cited above. Representative aminoprotecting groups for synthetic use include acyl groups such astert-butoxycarbonyl, benzyloxycarbonyl, benzoyl, acetyl and the like.Yet other conventional amino protecting groups for use in synthesis aredescribed in the literature, e.g. in the Bodanszky publication and Ivespatent referred to hereinabove.

As with the carboxyl protecting groups, when the amino protecting groupis intended to be present in a pharmaceutically useful peptide endproduct, then it will be selected from among amino protecting groupswhich offer low toxicity and the desired degree of lipophilicity andrate of in vivo cleavage. Especially suitable for in vivo use as aminoprotecting groups T are activated carbamates, i.e. the protecting groupT has the structure ##STR44## wherein R_(h) is hydrogen, C₁ -C₇ alkyl orphenyl and R₁ can be selected from the groups indicated as suitablecarboxyl protecting groups W hereinabove. Again, the bulkier groups arepreferred for use in vivo, and R₁ is preferably a polycycloalkyl orpolycycloalkenyl-containing group, such as adamantyl or a sterolresidue, especially a cholesterol or bile acid residue.

Alternatively, the amino protecting group T may simply be composed ofone or two "throw away" natural amino acids, e.g. alanine orphenylalanine, which are not needed for activity but which are designedto be cleaved first from the rest of the molecule by a specificpeptidase and in this way prevent premature degradation of the activeportion of the molecule.

The various protecting groups for hydroxyl, carboxyl and amino functionsdiscussed above can be substituted for the hydroxyl, carboxyl and aminofunctions in the instant amino acids/peptides (or their precursormolecules) by methods well-known in the art. Methods for chemicalremoval of the protecting groups (when such are not to be retained inthe pharmaceutically useful end product) are likewise well-known tothose skilled in the art. Typically, amine protecting groups arechemically removed by acidolysis (acid hydrolysis) or hydrogenation,depending on the particular protecting group employed. Hydroxyl andcarboxyl protecting groups are typically removed chemically by acid orbase hydrolysis. Protecting groups which are incorporated in thepharmaceutical end product must be amenable to hydrolytic or metaboliccleavage in vivo.

In general, the peptides provided by the present invention are preparedby sequential addition of one or more amino acids or protected aminoacids, with the redox system being either first incorporated into anamino acid or protected amino acid which is then added to the growingpeptide chain, or else inserted directly into a dipeptide or largerpeptide fragment in the course of the peptide synthesis. Methods forsequential addition of amino acids to form peptides, utilizingprotecting groups where appropriate, are well-known in the art. Anexcellent summary of such methods, including both solid phase synthesisand synthesis in solution, is contained in Nestor et al U.S. Pat. No.4,530,920, which is incorporated by reference herein in its entirety andrelied upon. See also SOLID PHASE PEPTIDE SYNTHESIS, second edition,John Morrow Stewart and Janis Dillaha Young, Pierce Chemical Company,Rockford, Illinois, 1984.

Peptides provided by the present invention can also be prepared bysegment condensation methods described in the literature, e.g. in theBodanszky and Houben-Weyl reference cited above.

The quaternary forms of the redox amino acids and peptides of thisinvention, i.e. the salts of formulas (II) and (B), respectively, can beprepared by a variety of methods suitably combined with theprotection/deprotection and peptides linkage steps referred to above.Typical methods of combining the various steps are illustrated in thesynthetic examples hereinafter. At the present time, three generalmethods have been found to be particularly convenient for preparation ofthe quaternary derivatives. Selection of an appropriate method dependsupon the identities of Z and R₁ in the desired products of formulas (II)and (B).

The first of the three methods for preparing the quaternary salts is ahalogen replacement reaction which is useful when Z in the desiredproduct of formula (II) or (B) is to be attached to the heterocyclicring via a ring nitrogen atom and R₁ is a direct bond. According to thismethod, a halogen-containing amino acid or peptide of the formula##STR45## respectively, wherein Hal is chloro or bromo and R₄, R₅ and Zare defined as before, is reacted with a compound of the formula##STR46## wherein R₂ and R₃ are defined as before. Preferred startingmaterials of formula (IV) include nicotinamide, picolinamide,isonicotinamide, 3-quinolinecarboxamide and 4-isoquinolinecarboxamide.The reaction is typically carried out in an inert organic solvent, withheating. The products of formulas (II) and (B) are of the type in whichZ is attached to the heterocyclic ring via a ring nitrogen atom, R₁ is adirect bond and X⁻ is a chloride or bromide anion.

The second of the three methods found useful in preparing the quaternarysalts of this invention is an alkylation reaction and can be used toprepare derivatives of formulas (II) and (B) wherein Z is attached tothe heterocyclic ring via a ring carbon atom and R₁ is C₁ -C₇ alkyl, C₁-C₇ haloalkyl or C₇ -C₁₂ aralkyl. In accord with this method, an aminoacid or peptide of the formula ##STR47## respectively, wherein R₂, R₃,R₄ and R₅ and Z are defined as before and Z is attached to theheterocyclic ring via a ring carbon atom, is reacted with an alkylatingagent of the formula

    R.sub.1 --halide                                           (VI)

wherein R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₂ aralkyl and thehalide is iodide, bromide or chloride. A preferred starting material offormula (VI) is methyl iodide. Typically, this quaternization reactiontakes place in a suitable organic solvent. The products of formulas (II)and (B) are of the type in which Z is attached to the heterocyclic ringvia a ring carbon atom, R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₂aralkyl and X⁻ is an iodide, bromide or chloride ion.

The third method for preparing the quaternary salts is a Zincke exchangereaction useful when Z in the desired product of formula (II) or (B) isto be attached to the heterocyclic ring via a ring nitrogen atom and R₁is a direct bond. Thus, a Zincke reagent of the formula ##STR48##wherein R₂ and R₃ are defined as before is reacted with an amino acid orpeptide having a pendant primary amino group, i.e. an amino acid/peptideof the formula ##STR49## respectively, wherein R₄, R₅ and Z are definedas before. This reaction is generally conducted in the presence of asuitable base, e.g. triethylamine, in an appropriate organic solvent,e.g. methanol, and affords derivatives of formulas (II) and (B) whereinZ is attached to the heterocyclic ring via a ring nitrogen atom, R₁ is adirect bond and X⁻ is a chloride anion. The Zincke reagent employed inthe process can be prepared by reacting 1-chloro-2,4-dinitrobenzene witha compound of formula (IV) above. Preferred Zincke reagents are thoseproduced from nicotinamide, picolinamide, isonicotinamide,3-quinolinecarboxamide and 4-isoquinolinecarboxamide. See also Zincke etal, Annalen, 1904, 333, 296; Lettre, Annalen, 1953, 579, 123; Keijzer etal, Heterocycles, Vol. 16, No. 10, 1981, 1687.

The various starting materials employed in the processes described aboveare commercially available or can be prepared by known methods.

When an anion is described which is different from the one obtained byone of the processes described above, the anion in the quaternary saltof formula (II) or (B) may be subjected to anion exchange via an anionexchange resin or, more conveniently, by use of the method of Kaminskiet al, Tetrahedron, Vol. 34, pp. 2857-2859 (1978). According to theKaminski et al method, a methanolic solution of an HX acid will reactwith a quaternary ammonium halide to produce the methyl halide and thequaternary .X salt.

The quaternary salts of formulas (II) and (B) can be reduced to form thecorresponding dihydro derivatives of formulas (I) and (A), respectively.Also, the quaternary salts of formulas (II) and (B) may be subjected tolengthening of the peptide chain, with reduction occurring at a laterpoint in the synthetic scheme. Frequently, it may be desirable topostpone reduction until the peptide chain has reached the desiredlength, i.e. the length desired for the pharmaceutical end product.

Reduction of the quaternary salts of formulas (II) and (B) to thecorresponding dihydro derivatives of formulas (I) and (A), respectively,it usually conducted at a temperature from about -10° C. to roomtemperature, for a period of time from about 10 minutes to 3 hours,conveniently at atmospheric pressure. The process is conducted in thepresence of a suitable reducing agent, preferably an alkali metaldithionite such as sodium dithionite, an alkali metal borohydride suchas sodium borohydride or lithium aluminum borohydride, or a morereactive dihydropyridine such as 1-benzyl-1,2-dihydroisonicotinamide.

Sodium dithionite reduction is conveniently carried out in an aqueoussolution, e.g. aqueous methylene chloride, in the presence of base e.g.sodium bicarbonate, and, in the case of pyridinium and quinoliniumstarting materials, generally affords a preponderance of 1,4-dihydroisomer. The dihydro product is usually insoluble in water and thus canbe readily separated from the sodium dithionite reaction medium.

In the case of sodium borohydride reduction, an organic reaction mediumis typically employed, e.g. a lower alkanol such as methanol, an aqueousalkanol or other protic solvent. For pyridinium and quinolinium startingmaterials, sodium borohydride reduction typically affords apreponderance of the 1,6-dihydropyridine and 1,2-dihydroquinolineisomers, respectively.

Other useful reducing agents include dihydropyridines which are morereactive than the quaternary salts which are to be reduced. Aparticularly suitable reagent of this type is the highly reactive1-benzyl-1,2-dihydroisonicotinamide, which can be used for selectivereduction of the quaternary salts by a direct hydride transfer reactionunder neutral conditions [Nuvole et al, J. Chem. Research, 1984, (S),356]. Thus, for example, pyridinium and quinolinium salts of theinvention can be regioselectively reduced to the corresponding1,4-dihydropyridines and 1,4-dihydroquinolines, respectively, utilizing1-benzyl-1,2-dihydroisonicotinamide as the reducing agent, typically ina suitable organic reaction medium, e.g. anhydrous methanol. Otherpossible reducing agents of the reactive dihydropyridine type includeribosyl N-methyl dihydronicotinamide (derived from NADH).

Especially preferred compounds of the present invention are the aminoacids of formula (I) in which the ##STR50## portion of the molecule hasone of the following structures, as well as the corresponding peptidesof formula (A) and the quaternary forms of these amino acids anddipeptides: ##STR51## In the above structures, Z is preferably --CH₂ --or --(CH₂)₄ --. In structures (e), (f), (g), (h), (i), (j) and (k), Z ismost preferably --(CH₂)--; in structures (c), (d), (l), (m) and (n), Zis most preferably --(CH₂)₄ --. The corresponding quaternary salts offormula (II) have the partial structures: ##STR52## wherein Z ispreferably as defined in the preceding two sentences.

As stated hereinabove, the novel amino acids of formulas (I) and (II)are useful in the preparation of novel bioactive peptides of the partialstructural formulas (A) and (B). In particular, by incorporating anamino acid fragment of structure (A) or (B) into the peptide chain,there is provided herein a method for imparting improved brainspecificity and substained in-brain activity to a pharmacologicallyactive peptide. The peptide thus modified is susceptible toadministration to a mammal in its reduced form comprising fragment (A)and is capable of in-brain conversion to its oxidized form comprisingfragment (B). The oxidized form either is itself pharmacologicallyactive or is convertible in vivo to a pharmacologically active form,e.g. by cleavage of one or more amino acid units. For most successfuluse in vivo, there are further incorporated into the final peptide (i.e.the peptide which is intended for administration) amino and carboxylprotective groups for the terminal amino and carboxyl functions of thepeptide, the protective groups being capable of in-brain cleavage tofree amino and carboxyl functions. Suitable such protective groups arediscussed hereinabove. The amino acid fragment of structure (A) or (B)is preferably located in a non-terminal position of the peptide chain;nevertheless, terminal positions are contemplated as well. Moreover,more than one redox fragment may be incorporated into a given peptide;multiple such fragments may be particularly desirable in the case oflarger peptides and/or when a peptide contains a plurality of suitablesites at which to introduce the redox fragment, i.e. a plurality oflocations at which the redox fragment can be placed without loss of thepeptide's pharmacological activity.

The final redox peptide of the invention preferably contains a total of2 to 20 amino acid units. Typically, except for the presence of at leastone redox amino acid fragment of structure (A) or (B) and the possibleprotection of terminal amino and carboxyl functions, the structure ofthe instant redox peptide is identical to that of a known, naturallyoccurring bioactive peptide or of a known bioactive synthetic peptide(particularly one which is an analog of a naturally occurring bioactivepeptide). Naturally, in order for the final redox peptide's brainspecificity/sustained activity to be of value, the peptide must exert auseful central activity, i.e. it should exert a significantpharmacological action in the central nervous system such that it may beused as a drug, that is, for the diagnosis, cure, mitigation, treatmentor prevention of disease or in the enhancement of desirable physical ormental development and conditions in man or animals, or it must beconvertible in vivo to a peptide having a useful central activity.

Appropriate known peptides for incorporation of the instant redox systemtherein include naturally occurring peptides such as kyotorphin, met⁵-enkephalin, leu⁵ -enkephalin, vasopressin, oxytocin, neurotensin, ACTHfragments, peptide T, Substance P. angiotensin, somatostatin and LH--RH,as well as their biologically active synthetic analogs. Many of thesepeptides and their analogs have been previously studied in depth, sothat it is possible to learn from the scientific literature whichpositions in the amino acid sequence are fairly critical to therequisite biological activity of a given peptide and which positions maybe varied without loss of activity. Some of these teachings of the artand their application to the instant invention are discussed below, butthis discussion is not intended to be exhaustive. In this discussion,the conventional peptide representation (amino terminus on left,carboxyl terminus on right) will be used, as will the conventionalabbreviations for the individual amino acid units (Phe forphenylalanine, Gly for glycine, etc.). Insofar as concernsconfiguration, in this general discussion the configuration of opticallyactive amino acids will be assumed to be L unless otherwise specified.

Kyotorphin is a dipeptide of the structure H-Tyr-Arg-OH which hasanalgesic properties. It has been found to stimulate the release ofenkephalin. The corresponding dipeptides in which one or both aminoacids has/have the D-configuration also have activity.

The enkephalins are two naturally occurring pentapeptides belonging tothe endorphin class. Met⁵ -enkephalin has the structure

    H-Tyr-Gly-Gly-Phe-Met-OH

while Leu⁵ -enkephalin has the structure

    H-Tyr-Gly-Gly-Phe-Leu-OH.

The most important property of the enkephalins is their morphine-likeanalgesic action. They also have a variety of effects on memory.

Some peptides slightly larger than the enkephalins with intrinsicactivity have also been identified. These include Met⁵ -enkephalin-Arg⁶and Met⁵ -enkephalin-Lys⁶, which are believed to be potential precursorsof Met⁵ -enkephalin; and Met⁵ -enkephalin-Arg⁶ -Phe⁷, which has highaffinity for K-opiate receptors.

Moreover, many enkephalin analogs have been synthesized (e.g. FK-33-824,LY 127623/metkephamid, Wy-42,896) and structure/activity relationshipshave been analyzed. See, in particular, J. S. Morley, Annu. Rev.Pharmacol, 20: 81-110 (1980), incorporated by reference herein in itsentirety and relied upon. While virtually every position in theenkephalin chain allows some variation without loss of activity, somepositions allow much more variation than others. Thus, in the case ofN-terminal substitution, activity can be maintained in the same range byaddition of an L-amino acid. The Gly² position appears to beparticularly amenable to variation, and replacement with a D-amino acidoften has been found to lead to a marked increase in potency and/orlonger biological half-life. Also, structural/conformational changes atthe Met⁵ /Leu⁵ position, i.e. replacing Met⁵ or Leu⁵ with a differentamino acid (L or D), have afforded analogs which are invariably active,although increases in potency as the result of such changes are modest.Replacement of the terminal acid function with an unsubstituted amidegroup has afforded a very potent analog, Met⁵ -enkephalin-NH₂. Inaddition, contraction or extension at the C-terminus has afforded activeanalogs.

Vasopressin and oxytocin are cyclic peptides which differ from eachother in only two amino acids. All mammalian oxytocin (OXT) has thestructure ##STR53## Most vasopressin is arginine-vasopressin (AVP),which has the structure ##STR54## swine vasopressin (SVP) is also knownas lysine-vasopressin and has the structure ##STR55## Vasopressinappears to enhance retention of learned responses and to enhanceattention and memory. Both oxytocin and vasopressin may be involved inpain mechanisms. In vasopressin, residues 2, 3 and 5 seem to be fairlycritical for activity, especially asparagine at 5. Most of the activityin vasopressin and oxytocin seems to be in the covalent ring structure.Removing the C-terminal glycinamide appears not to affect behavior.Behaviorally active fragments include H-Pro-Arg-Gly-NH₂, H-Lys-Gly-NH₂,AVP₁₋₇ (pressinamide), OXT₁₋₈, OXT₁₋₆, Pro-Leu-Gly-NH₂ and Leu-Gly-NH₂.While the amino acids at 8 and 9 can be removed, shortening to 6 or 7amino acids appears to cause a change in activity, at least in the caseof oxytocin, where OXT₁₋₇ and OXT₁₋₆ affect memory differently from OXT.

Neurotensin (NT) is a basic tridecapeptide of the formula

    p-Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH

which has a variety of hormone-like activities. It has been shown toinduce hypotension. It also acts as a CNS neurotransmitter and appearsto be a very potent analgesic. The carboxy terminal leucine moietyappears to be essential for binding and the arginine residues at 8 and 9also are essential for binding and biological activity. Very littlevariation as positions 11, 12 and 13 seems to be possible. Xenopsin, anoctapeptide which shares many properties with neurotensin, has thestructure

    pGlu-Gly-Lys-Arg-Pro-Trp-Ile-leu-OH.

Other potent neurotensin analogs include D-Tyr¹¹ -NT and D-Phe¹¹ -NT.

ACTH, or adrenocorticotropic hormone, has complex behavioral activitiesinvolving learning, memory, motivation, arousal and attention. It has 39amino acids, but its essential structure is believed to be ACTH₄₋₇, withphenylalanine in position 7 playing a key role in behavioral effects.Human ACTH₁₋₃₉ has the structure: ##STR56## Active fragments includeACTH₄₋₁₀ and ACTH₄₋₇, with ACTH₄₋₇ being the shortest peptide found togive the typical behavioral effects of ACTH. A very active ACTH₄₋₉analog, OGR 2766, has the structure

    H-Met(O)-Glu-His-Phe-D-Lys-Phe-OH,

which has an oxidized methionine at 4, D-lysine at 8 in place ofarginine, and phenylalanine at 9 in place of tryptophan. It shows 1000fold potentiation, with dissociation of behavioral effects fromendocrine, metabolic and opiate-like activities. Other active analogsinclude D-Phe⁷ -ACTH₁₋₁₀, D-Phe⁷ -ACTH₄₋₇ and D-Phe⁷ -ACTH₄₋₁₀, althoughin some ways these analogs behave oppositely from the natural L-forms.

Peptide T is an octapeptide with anti-AIDS activity. Substance P is anundecapeptide which acts as a vasodilator and a depressant, and canproduce analgesia and hyperalgesia. It plays an important role innervous system function. Substance P has the structure ##STR57## Itappears that most structural variation can occur at positions 1 to 6,with some variation also possible at 8. Active analogs includephysalaemin, which has the structure

    pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH.sub.2 ;

eledoisin, which has the structure

    pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH.sub.2 ;

and kossinin, which has the structure

    Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH.sub.2.

Moreover, a series of retro-inverso C-terminal hexapeptide analogs ofSubstance P has been recently described by Verdini et al, U.S. Pat. No.4,638,046, dated Jan. 20, 1987, incorporated by reference herein andrelied upon. Verdini et al describe compounds of the formula ##STR58##in which

P is a hydrogen atom, a linear or branched aliphatic alkyl group with1-6 carbon atoms, or a saturated or unsaturated linear or branched chainaliphatic acyl group such as formyl, acetyl, propionyl, n-butyryl,isobutyryl, n-valeryl, isovaleryl, hexanoyl, isohexanoyl, heptanoyl,octanoyl, crotonoyl, methacryloyl, acryloyl; or a substituted acyl groupsuch as hydroxyacetyl, 2-hydroxypropionyl, 3-hydroxypropionyl,aminoacetyl, 4-hydroxyphenylacetyl, 4-hydroxyphenylpropionyl,2-aminopropionyl, 3-aminopropionyl, O-ethylmalonyl, ethoxyformyl,methoxyacetyl, 3-methoxypropionyl, 3-ethoxypropionyl, chloroacetyl,dichloroacetyl, 2-chloropropionyl, 3-chloropropionyl,2,3-dichloropropionyl, bromoacetyl, 4-hydroxy-3,5-diiodophenylacetyl,3-oxobutyryl, 3-oxovaleryl, 4-oxovaleryl, methylthioacetyl,3-methylthiopropionyl, ethylthioacetyl, 3-ethylthiopropionyl,nicotinoyl, 4-aminobutyryl, B.sup.α-[(1-(9-adenyl)-β-D-ribofuranuronosyl)], N.sup.α[(1-(9-hypoxanthyl)-β-D-ribofuranuronosyl]; or a group such asbenzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl,isobornyloxycarbonyl, adamantyloxycarbonyl, or chloro ornitro-substituted benzyloxycarbonyl;

R¹ is a residue of methionine, methionine sulphoxide, methioninesulphone, selenomethionine, leucine, norleucine, valine or norvaline;

R² is a residue of leucine, norleucine, valine, norvaline, alanine orisoleucine;

R₃ is a hydrogen atom or methyl;

R⁴ is an amino acid residue of D configuration such as phenylalanine,tryptophan, tyrosine, valine, norvaline, leucine, norleucine,isoleucine, serine or derivatives, threonine or derivatives, histidineor derivatives, methionine, methionine-S-methyl, methionine sulphone,arginine or derivatives, lysine or derivatives, ornithine orderivatives, 2,4-diaminobutyric acid or derivatives,2,3-diaminopropionic acid or derivatives, glutamic acid or aspartic acidor their suitable derivatives:

R⁵ is a hydrogen atom or the side-chain of amino acids such asphenylalanine, tyrosine, 4-chlorophenylalanine, O-benzyltyrosine (ortheir acetyl, cyclopentyl, tert-butyloxycarbonyl or4-hydroxyphenylacetyl derivatives);

R⁶ is an amino acid residue such as glutamine or derivatives,pyroglutamic acid, alanine, tyrosine, lysine or derivatives, proline,N-formyl-proline, β-alanine, N-acetyl-β-alanine, glycine,desaminophenylalanine, desaminoaspartic acid, methyldesaminoasparticacid, or glutamic acid esters represented by the general formula##STR59## in which X is methyl, ethyl, methoxyethyl ormethoxy(ethoxy)_(n) ethyl wherein n=1, 2 or 3.

The Verdini et al analogs show variations possible in the amino acidscorresponding to positions 6, 7 and 9-11 of Substance P, substantialvariations at position 8 of Substance P (R⁴ of Verdini et al) using awide variety of D amino acid residues, and the ability to delete thefirst five amino acids in Substance P without loss of activity.

A group of biologically active heptapeptides has been recently describedby deCastiglione et al, U.S. Pat. No. 4,567,162, dated Jan. 28, 1986,incorporated by reference herein and relied upon. These peptides aresaid to display activity on the central nervous system and to be usefulin promoting growth activity and improving feed efficiency in animals.The compounds have the general formula

    X-Val-Pro-Pro-Leu-Gly-Trp-A-Y

wherein:

X represents a hydrogen atom or a terminal nitrogen protecting group ofacyl, aliphatic urethane, aromatic urethane, alkyl or aralkyl type;

A represents a neutral L-α-amino acid residue; and

Y represents a hydroxy group, an amino group or a group of the formulaOR, NHR, NR₂ or NH--N--H--R' wherein R represents a straight chain,branched chain or cyclic (including fused or bridged rings) alkyl grouphaving up to 11 carbon atoms which may be unsubstituted or independentlysubstituted by a hydroxy or amino group or a halogen atom, an aralkylgroup having from 7 to 14 carbon atoms or a phenyl group; and R'represents a hydrogen atom, any of the groups which B may represent, astraight chain, branched chain or cyclic aliphatic acyl group havingfrom 1 to 11 carbon atoms which may be unsubstituted or independentlysubstituted by a hydroxy or an amino group or a halogen atom, anaromatic acyl group which may be unsubstituted or independentlysubstituted by a hydroxy or amino group or a halogen atom, a straightchain, branched chain or cyclic aliphatic urethane type group havingfrom 3 to 11 carbon atoms, or an aromatic urethane type group. Thus, asignificant amount of variation is possible in the structure of theseventh, or terminal amino acid, A.

Angiotensin is a pressor substance. Angiotensin I, a decapeptide, isconverted to the active pressor agent, angiotensin II, by splitting offthe C-terminal His-Leu residues. The octapeptide II differs amongspecies at positions 5 (Val or Ile). The active angiotensin II as wellas angiotensin amide are hypotensive and may increase the effectivenessof endogenous norepinephrine. These peptides have the followingstructures: ##STR60## Thus, it would appear that at least the C-terminaldipeptide portion of Angiotensin I could be modified without loss ofactivity.

Somatostatin (SRIF) is a cyclic tetradecapeptide that with GRF mediatesthe neuroregulation of pituitary growth hormone release. It is also apotent inhibitor of central and peripheral neural systems. It has beenused as an experimental hypoglycemic and growth hormone inhibitor.Somatostatin has the formula ##STR61## Potent peptides having anN-terminal extension of SRIF-14 have also been isolated. It is believedthat modification of the non-cyclic portion of somatostatin, byreplacing part or all of the N-terminal dipeptide or by adding to theN-terminus, can be undertaken without loss of activity.

LH--RH, or GnRH, is the luteinizing hormone-releasing factor. It is theneurohumoral hormone produced in the hypothalamus which stimulatessecretion of LH and FSH, which in turn regulate functioning of thegonads (by stimulating production of steroid hormones) and regulategamete production and maturation. LH--RH is a decapeptide having thestructural formula

    p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2.

Agonist analogs of LH--RH may be used to control fertility in twodifferent ways. Thus, low doses of LH--RH analogs can be used tostimulate ovulation in the female as well as spermatogenesis andandrogen production in the male. Larger doses of LH--RH analogs,especially long-acting, highly potent analogs, paradoxically blockovulation and suppress spermatogenesis. In domestic animals, the lattereffect promotes weight gain and generally acts as a sterilant.Antagonist analogs of LH--RH, i.e. analogs which are antagonistic to thenormal function of L--RH, may be used as male or female contraceptives,in the treatment of endometriosis and precocious puberty in females andin the treatment of prostatic hypertrophy in males. Basically, theantagonist analogs are used to inhibit the production of gonadotropinsand sex hormones, which is essentially the same as the high dose,paradoxical effect of the agonist analogs.

It is now well-known that the glycine residue in the 6-position ofLH--RH can be replaced by a variety of D-amino acids to give LH--RHagonists and antagonists of much greater potency than the naturalhormone itself. Other changes which have resulted in substantialincrease or retention of activity include eliminating Gly-NH₂ at the10-position to give a nonapeptide as an alkyl-, cycloalkyl- orfluoroalkylamide; replacing Gly-NH₂ at the 10-position with anα-azaglycine amide; replacing Leu at the 7-position withN-methyl-leucine; replacing Trp at the 3-position with3-(1-naphthyl)-L-alanyl or with 3-(2-napthyl)-L-alanyl; and replacingTyr at the 5-position with phenylalanyl or with3-(1-pentafluorophenyl)-L-alanyl.

Numerous LH--RH analogs, most frequently nonapeptides and decapeptides,have been developed to date. For example, Nestor et al, U.S. Pat. No.4,530,920, dated July 23, 1985, incorporated by reference herein in itsentirety and relied upon, provides nonapeptide and decapeptide agonistanalogs of LH--RH which have the formula ##STR62## and thepharmaceutically acceptable salts thereof, wherein:

A is tryptophyl, phenylalanyl, 3-(1-napthyl)-L-alanyl or3-(2-naphthyl)-L-alanyl;

B is tyrosyl, phenylalanyl or 3-(1-pentafluorophenyl)-L-alanyl;

C is an amino acyl residue selected from the group consisting of theradicals represented by the following structural formulas: ##STR63##wherein

n is 1 to 5;

R₁ is alkyl of 1 to 12 carbon atoms, --NRR₃ wherein R is hydrogen oralkyl of 1 to 4 carbon atoms, R₃ is hydrogen, alkyl of 1 to 12 carbonatoms, cycloalkyl, fluoroalkyl, phenyl, benzyl, --(CH₂)_(n) --morpholinoor --(CH₂)_(n) N(R₄)₂ wherein n is 1 to 5 and R₄ is lower alkyl;

R₂ is hydrogen or R₃ ; or R₁ and R₂ comprise a ring represented by thefollowing structural formulas: ##STR64## wherein n is 1 to 7; A ishydrogen, alkyl of 1 to 6 carbon atoms or cycloalkyl; and X is halo orA; or ##STR65## wherein R₅ is alkyl of 1 to 6 carbon atoms, benzyl,phenylethyl, cyclohexyl or cyclopentyl; R₆, R₇ and R₈ are hydrogen oralkyl of 1 to 4 carbon atoms; and n is the integer 2-5; or ##STR66##wherein

R₉ is hydrogen, alkyl of 1 to 12 carbon atoms, phenyl orphenylloweralkyl;

D is leucyl, isoleucyl, not-leucyl, N-methylleucyl or tryptophanyl;

E is arginyl or leucyl; and

F is glycinamide or --NH--R¹, wherein

R¹ is lower alkyl, cycloalkyl, fluoro lower alkyl or --NH--CO--NH--R²wherein R² is hydrogen or lower alkyl.

Exemplary antagonist analogs of LH--RH are provided by Rivier et al U.S.Pat. No. 4,565,804, dated Jan. 21, 1986 and Rivier et al U.S. Pat. No.4,569,927, dated Feb. 11, 1986, both incorporated by reference herein intheir entirety and relied upon. The '804 peptides have the structure

    X-R.sub.1 -(W)D-Phe-R.sub.3 -R.sub.4 -R.sub.5 -R.sub.6 (V)-R.sub.7 -Arg-Pro-R.sub.10

wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, Pro, D-pGlu, D-Phe, D-Trp or β-D-NAL; W is F, Cl, Cl₂Br, NO₂ or C.sup.α MeCl; R₃ is D-Trp, (N^(in) For)D-Trp or D-Trp whichis substituted in the 5- or 6-position with NO₂, NH₂, OCH₃, F, Cl, Br orCH₃ ; R₄ is Ser, Orn, AAL or αBu; R₅ is Tyr, (3F)Phe, (2F)Phe, (3I)Tyr,(3CH₃)Phe, (2CH₃)Phe, (3Cl)Phe or (2Cl)Phe; R₆ is D-Lys, D-Orn or D-Dap;V is arg-R',R")n(X), with n being 1 to 5 and R' and R" being H, methyl,ethyl, propyl or butyl; R₇ is Leu, NML, Nle or Nva; and R₁₀ is Gly-NH₂,D-Ala-NH₂ or NH-Y, with Y being lower alkyl, cycloalkyl, fluoro loweralkyl or ##STR67## where Q is H or lower alkyl.

In the '804 peptides, the expression "β-D-NAL" means the D-isomer ofalanine, substituted by naphthyl on the β-carbon, or 3-D-NAL.Preferably, the β-carbon is attached at the 2-position or naphthalene(β-D-2NAL), but β-D-1NAL may also be used. "(C.sup.α Me-4Cl)Phe" meansphenylalanine substituted at the para position with chloro, the α-carbonbeing methylated. "Dap" means α,β-diaminopropionic acid, orβ-aminoalanine. "NML" means N.sup.α CH₃ -L-Leu. "AAL" means β-amino-Alaor Dap, and "aBu" means α,γ-diaminobutyric acid, either of which or Ornmay be present in the 4-position. DehydroPro is preferably at position 1when Ser is not present at position 4. "R₆ (arg-R',R")n(X)" means theD-amino acid in the main chain, which through its side chain aminofunction also forms part of the arginine-containing peptide side chain.

The '927 peptides have the structure

    X-R.sub.1 -(W)D-Phe-R.sub.3 -R.sub.4 -R.sub.5 -R.sub.6 -R.sub.7 -Arg-Pro-R.sub.10

wherein x is hydrogen or an acyl group having 7 or less carbon atoms; R₁is dehydro-Pro, D-pGlu, D-Phe, D-Trp or β-D-NAL; W is F, Cl, Cl₂ Br, NO₂or C.sup.α MeCl; R₃ is (N^(in) For)D-Trp or D-Trp which is substitutedin the 5-or 6-position with NO₂, NH₂, OCH₃, F, Cl, Br or CH₃ ; R₄ isSer, Orn, AAL or aBu; R₅ is Tyr, Arg, (3F)Phe, (2F)Phe, (3I)Tyr,(3CH₃)Phe, (2CH₃)Phe, (3Cl)Phe or (2Cl)Phe; R₆ is A(4NH₂)D-Phe, D-Lys,D-Orn, D-Har, D-His, (4gua)D-Phe, D-Tyr, a D-isomer of a lipophilicamino or D-Arg; R₇ is Leu, NML, Nle or Nva; and R₁₀ is Gly-NH₂,D-Ala-NH₂ or NH-Y, with Y being lower alkyl, cycloalkyl, fluoro loweralkyl or ##STR68## where Q is H or lower alkyl, provided however thatwhen R₅ is Arg, R₆ is D-Tyr. The various terms are as defined with the'804 peptides.

It is clear from the Nestor et al and Rivier et al patents that theamino acid in the 6-position of the LH-RH peptide chain is very amenableto replacement by numerous D-amino acids, including unnatural aminoacids which have sizable side chains. These patents also confirm theextent of other permissible structural changes discussed hereinabove forLH--RH analogs, e.g. at the 3-, 5- and 7-positions.

It will be apparent from the foregoing that many peptides offer amultiplicity of locations at which a redox amino acid fragment of thepresent invention might be inserted without loss of the peptide'scharacteristic activity. Nevertheless, it is preferred that the redoxsystem be located in a position which allows a great deal of structuralvariation without loss of activity and/or that the specific redoxfragment selected be isosteric (or approximately so) with the amino acidunit which it is intended to replace. At the present time, the followingredox amino acid fragments are preferred specific embodiments of thisinvention:

    __________________________________________________________________________    REDOX AMINO ACID FRAGMENT                 ABBREVIATION                        __________________________________________________________________________     ##STR69##                           Pyrala                                    ##STR70##                           Quinala                                   ##STR71##                           Pyrlys                                   __________________________________________________________________________

However, it is to be understood that these particular redox fragmentsare intended to be illustrative, not limitative, of the instantfragments which may be incorporated into known centrally activepeptides.

Some especially preferred embodiments of the present invention are thefollowing:

(a) Enkephalin analogs of the structures

    H-Tyr-(A/B)-Gly-Phe-Met-OH

and

    H-Tyr-(A/B)-Gly-Phe-Leu-OH

and the corresponding terminal amino protected and terminal carboxylprotected compounds (including the corresponding C-terminal amides) inwhich the protecting groups are as defined hereinabove and (A/B)represents a redox amino acid fragment of structure (A) or (B)hereinabove, especially when (A/B) is Pyrala, Quinala or Pyrlys, havingthe L- or D-configuration;

(b) Enkephalin analogs of the structure

    H-Tyr-Gly-Gly-Phe(A/B)-OH

and the protected counterparts as in (a) above, especially when (A/B) isPyrala, Quinala or Pyrlys, having the L- or D-configuration;

(c) Vasopressin and oxytocin analogs in which the amino acids at the 8-and/or 9-positions are replaced with L- or D-(A/B) as defined above,especially when (A/B) is Pyrala, Quinala or Pyrlys;

(d) Neurotensin analogs in which Lys⁶ or Tyr¹¹ is replaced with L- orD-(A/B) above, and their C-terminal protected counterparts, especiallythose in which Pyrlys is in the 6-position or Pyrala is in the11-position;

(e) ACTH₁₋₁₀, ACTH₄₋₇ and ACTH₄₋₁₀ analogs in which Phe⁷ is replacedwith D-(A/B), and their protected counterparts, especially when (A/B) isPyrala;

(f) ACTH₄₋₉ analogs of the structures

    H-Met(O)-Glu-His-Phe-D-(A/B)-Phe-OH

    and

    -Met(O)-Glu-His-Phe-D-Lys-(A/B)-OH

and their protected counterparts, especially those of the firststructure where D-(A/B) is D-Pyrlys and those of the second where (A/B)is Pyrala or Quinala;

(g) ACTH₁₋₁₀ wherein Trp⁹ is replaced with (A/B) and its protectedcounterparts, especially when (A/B) is Pyrala or Quinala;

(h) ACTH₄₋₁₀ wherein Arg⁸ is replaced with D-(A/B) and its protectedcounterparts, especially when (A/B) is Pyrala or Quinala;

(i) Substance P analogs in which Phe⁸ is replaced with (A/B) and theirN-terminal protected counterparts, especially when (A/B) is Pyrala;

(j) Substance P analogs in which any one of the amino acids at positions1 to 6 is replaced with (A/B), and their N-terminal protectedcounterparts, especially when (A/B) is Pyrala, Quinala or Pyrlys;

(k) Hexapeptide analogs of Substance P having the structure defined inVerdini et al U.S. Pat. No. 4,638,046 and set forth hereinabove, exceptthat Verdini et al's R⁴ amino acid residue is replaced with an aminoacid fragment of the present invention D-(A/B), especially D-Pyrala,D-Quinala or D-Pyrlys;

(l) Heptapeptide analogs of the growth promoting peptides defined indeCastiglione et al U.S. Pat. No. 4,567,162 and set forth hereinabove,except that deCastiglione et al's amino acid unit A is replaced with anamino acid fragment of the present invention L-(A/B), especiallyL-Pyrala, L-Quinala or L-Pyrlys;

(m) Angiotensin I analogs in which one or both of the amino acids in theC-terminal dipeptide portion is replaced with (A/B), and the protectedcounterparts (especially the N-terminal amide), particularly when (A/B)is Pyrala, Quinala or Pyrlys;

(n) Somatostatin analogs in which (A/B) replaces part or all of theN-terminal dipeptide portion or in which (A/B) is added to theN-terminus, and the corresponding terminal amino protected derivatives,especially when (A/B) is Pyrala, Quinala or Pyrlys;

(o) LH--RH analogs having the structure defined in Nestor et al U.S.Pat. No. 4,530,920 and set forth hereinabove, except that Nestor et al'samino acid residue C in the 6-position is replaced with an amino acidfragment of the present invention (A/B), especially D-(A/B), where (A/B)is preferably Pyrala, Quinala or Pyrlys; and

(p) LH--RH analogs of the structures depicted in the Rivier et al '804and '927 patents and set forth hereinabove, except that Rivier et al'samino acid residue in the 6-position is replaced with an amino acidfragment of the present invention D-(A/B), especially when (A/B) isPyrala, Quinala or Pyrlys.

In instances in which isosteric considerations are important, typicallya fragment of the instant invention such as Pyrala will be used toreplace phenylalanine or tyrosine, a fragment such as Quinala will beused to replace tryptophan and a fragment such as Pyrlys will be used toreplace lysine, arginine or histidine in a peptide chain.

The methods for synthesizing the amino acids and peptides of the presentinvention have already been discussed hereinabove. In some cases, themethods of peptide synthesis can be simplified by utilizing commerciallyavailable bioactive peptides and their fragments, SIGMA® CHEMICALCOMPANY, Post Office box 14508, St. Louis, Mo. 63178 U.S. has a largenumber of such products available; some of those which may be useful inpreparing the instant redox peptides include, for example, angiotensinII, neurotensin fragment 1-6, neurotensin fragment 1-8, neurotensinfragment 8-13, somatostatin, Substance P fragment 1-4, Substance Pfragment 4-11, Substance P fragment 5-11 and Substance P fragment 7-11.

In order to further illustrate the amino acids and peptides of thepresent invention, the following synthetic examples are given, it beingunderstood that same are intended only as illustrative, as manymodifications in materials and methods will be apparent to those skilledin the art.

In the examples to follow, all melting points were taken on a Mel-Tempapparatus and are not corrected. Elemental analyses were performed atAtlantic Microlabs, Inc., Atlanta, Ga.

EXAMPLE 1 Preparation of N-(tert-Butoxycarbonyl)-3-chloro-L-ala-nine

To a solution of 1.0 g (6.25 mmol) of 3-chloro-L-alanine and 1.3 mL oftriethylamine in 50% aqueous 1,4-dioxane were added 1.69 g (6.90 mmol)of 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile with stirring atroom temperature. After 2 hours, the reaction mixture was poured into 20mL of water and extracted twice with ethyl acetate. The organic layerwas washed twice with water and then with saturated brine, dried overanhydrous magnesium sulfate and evaporated to give 0.66 g (40.5%) ofwhite solid. Recrystallization from a mixture of ethyl acetate andn-hexane afforded 0.58 g of the pure product (35.6%) melting at 123-124°C., with decomposition, and having the structural formula: ##STR72## Theidentity of the product was confirmed by IR and NMR analyses.

EXAMPLE 2 Preparation ofN-(tert-Butoxycarbonyl)-3-chloro-L-ala-nylglycine ethyl ester

To a solution of 0.28 g (2 mmol) of glycine ethyl ester hydrochlorideand 0.28 mL (2 mmol) of triethylamine in 10 mL of chloroform, 0.52 g (2mmol) of N-(tert-butoxycarbonyl)-3-chloro-L-alanine in 6 mL ofchloroform and 0.42 g (2 mmol) of dicyclohexylcarbodiimide were added at0° C. The reaction mixture was stirred for 16 hours at room temperature.The resultant precipitate was removed by filtration and the filtrate waswashed successively with 0.5N hydrochloric acid, water and 3% aqueoussodium bicarbonate, then was dried over anhydrous sodium sulfate.Removal of the solvent left a crystalline residue which wasrecrystallized from a mixture of ethyl acetate and n-hexane to give0.415 g (60%) of the pure product melting at 71.5-72.0° C. The identityof the product, which has the structural formula ##STR73## was confirmedby IR and NMR analyses.

EXAMPLE 3 Preparation ofL-1-[2-(tert-Butoxycarbonyl)amino-2-(N-ethoxycarbonylmethyl)carbamoyl]ethyl-3-carbamoylpyridiniumchloride

A solution of 0.11 g (0.32 mmol) ofN-(tert-butoxycarbonyl)-3-chloro-L-alanylglycine ethyl ester and 47 mg(0.39 mmol) of nicotinamide in 2 mL of dry acetone was gently refluxedovernight. During reflux, acetone was removed by evaporation to leave apale brown solid. This procedure was repeated three times. Then thewell-dried solid was washed five times with 1 mL portions of dry acetoneto give 50 mg (27.5%) of pale brown solid. Recrystallization from amixture of methanol and acetonitrile gave 16 mg of pale brown crystals,melting at 223-235° C. The identity of the product, which has thestructural formula ##STR74## was further confirmed by IR analysis.

EXAMPLE 4 Preparation ofN-(tert-butoxycarbonyl)-3-chloro-L-alanyl-L-phenylalanyl-L-leucine ethylester

To a solution of 0.42 g (1 mmol) of L-phenylalanyl-L-leucine ethyl estertrifluoroacetate, 0.14 mL (1 mmol) of triethylamine and 0.26 g (1 mmol)of N-(tert-butoxycarbonyl)-3-chloro-L-alanine in 8 mL of chloroform,0.21 g (1 mmol) of dicyclohexylcarbodiimide was added at 0° C. Theresultant reaction mixture was stirred overnight at room temperature.The precipitate which formed was removed by filtration and the filtratewas washed successively with 0.5 N hydrochloric acid, water, 3% aqueoussodium bicarbonate and water, then dried over anhydrous sodium sulfate.Removal of the solvent afforded a white solid, which was mixed with 4 mLof a 1:1 mixture of ethyl acetate and n-hexane. Insoluble materials wereremoved by filtration and the filtrate was evaporated. The residue wasrecrystallized from a mixture of ethyl acetate and n-hexane to give0.367 g (66.5%) of the desired product. The product, which has thestructural formula ##STR75## melted at about 110° C. Its identity wasfurther confirmed by IR and NMR analyses and by thin layerchromatography (R_(f) =0.34 in 5:1 chloroform/ethyl acetate).

EXAMPLE 5 Preparation of N-Benzoyl-3-(3-quinolyl)alanine methyl ester

A mixture of 1.46 g (9.3 mmol) of 3-quinolinecarboxaldehyde, 1.8 g (10mmol) of hippuric acid, 0.6 g (7.0 mmol) of anhydrous sodium acetate and8 mL of acetic anhydride was heated gradually and stirred for 30 minutesat 90°-100° C. To the hot reaction mixture were added 10 mL of hotwater. The reaction mixture was then allowed to cool to roomtemperature. The yellow solid which formed was removed by filtration,washed once with 50% aqueous acetic acid and twice with ethanol, thendried in vacuo to give 2.39 g of yellow solid melting at 200°-202° C.Recrystallization from a mixture of chloroform and ethyl acetateafforded 2.05 g (73.4%) of the pure azolactone melting at 209.5°-210° C.and having the structural formula: ##STR76## IR and NMR analysesconfirmed the identity of the azolactone.

A mixture of 2.0 g (67 mmol) of the azolactone and 0.3 g of 10%palladium on carbon catalyst in 60 mL of acetic acid was vigorouslystirred under hydrogen overnight. The catalyst was removed by filtrationand washed thoroughly with dimethylformamide. The filtrate and washingwere combined and concentrated. The residue was hydrogenated overnightusing 0.3 g of 10% palladium on carbon as the catalyst. The catalyst wasremoved by filtration and the filtrate was evaporated to give a reddishbrown solid. The solid was suspended in dry methanol; then, hydrogenchloride gas was passed through the suspension, with cooling in icewater, until saturation was reached. The reaction mixture was allowed tostand overnight, then was evaporated. The resultant residue wasneutralized with a sufficient amount of aqueous sodium bicarbonate andextracted twice with chloroform. The combined organic layers were washedwith water, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was chromatographed on silicon (IV) oxide,using 2% methanol in chloroform as eluent, to give 0.41 g of an oilwhich solidified on standing. Recrystallization from a mixture of ethylacetate and isopropyl ether gave 0.25 g of pale brown crystals meltingat 124°-125° C. The identity of the product, which has the structuralformula ##STR77## was confirmed by IR analysis.

EXAMPLE 6 Preparation of3-(2-Benzoylamino-2-methoxycarbonyl)-ethyl-1-methylquinolinium iodide

A solution of 0.21 g (0.63 mmol) of N-benzoyl-3-(3-quinolyl)alaninemethyl ester and 1.0 mL of iodomethane in 5.0 mL of anhydrous acetonewas refluxed overnight. The resulting precipitate was removed byfiltration and recrystallized twice from a mixture of acetonitrile andacetone to give yellow crystals, which were dried in vacuo at 80° C. toafford 0.21 g (40%) of orange crystals melting at 110°-114° C. Theproduct, which was further characterized by IR and NMR analyses, has thestructural formula: ##STR78##

EXAMPLE 7 Preparation of N.sup.ε -Benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysine methyl ester

An ethyl solution of 1.48 g (3.9 mmol) of N.sup.ε-benzyloxycarbonyl-N.sup.α -(tert-butoxycarbonyl)-L-lysine was combinedwith an ethyl ether solution of diazomethane at 0° C. The reactionmixture was evaporated to give a colorless oil in nearly quantitativeyield (1.54 g). TLC R_(f) =0.38 (chloroform/ethyl acetate, 5:1). IR(neat) ν 3340, 1710, 1530 cm⁻¹. NMR (CDCl₃) δ 7.3 (5H, s), 7.25 (1H, m),5.05 (2H, s), 5.0 (1H, m), 4.1 (1H, m), 3.8 (3H, s), 3.1 (2H, m),2.0-1.1 (6H, m), 1.4 (9H, s). The product has the structural formula:##STR79##

EXAMPLE 8 Preparation of 3-Carbamoyl-1-(2,4-dinitrophenyl)pryidiniumchloride

A mixture of 20 g (98.7 mmol) of 1-chloro-2,4-dinitrobenzene and 8 g(65.5 mmol) of nicotinamide was gradually heated to 100° C., thenstirred at that temperature for one hour. The mixture was cooled toambient temperature and 100 mL of anhydrous methanol were added. Theresultant solution was poured into 400 mL of anhydrous ethyl ether, withvigorous stirring. The solvent was removed by decantation and theresidual precipitate was dissolved in 100 mL of methanol. The resultantsolution was poured into 400 mL of anhydrous ethyl ether, the solventwas again removed by decantation and the solid residue was dissolved in200 mL of water. The aqueous solution was refluxed with active charcoalfor 30 minutes, then filtered. The filtrate was evaporated and dried invacuo over phosphorus pentoxide to give 14.7 g (69.2%) of the titlecompound of the formula ##STR80##

EXAMPLE 9 Preparation ofL-1-[5-(tert-butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoylpyridiniumchloride

A mixture of 0.69 g (1.75 mmol) of N.sup.ε -benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysine methyl ester and 0.17 g of 10% palladiumon carbon catalyst in 10 mL of methanol was stirred under a hydrogenatmosphere for 3 hours. The catalyst was then removed by filtration. Tothe filtrate was added a solution of 0.568 g (1.75 mmol) of3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride in 1.5 mL ofmethanol. The resultant reaction mixture was refluxed overnight, thenthe precipitate was removed by filtration and the filtrate wasevaporated. The residual oil was triturated with a mixture of acetoneand ethyl acetate, affording a yellow solid. The solvent was removed andthe residue was washed with a mixture of acetone and ethyl acetate anddried in vacuo to give 0.308 g (45.6%) of the title compound as a paleyellow solid. The product, which has the structure ##STR81## wascharacterized as follows: IR(KBr tablet) ν 3380, 1730, 1695, 1685, 1510,1390, 1165 cm⁻¹ ; NMR (DMSO-d₆) δ 9.83 (1H, s), 9.33 (1H, d, J=6 Hz),9.1 (2H, m), 8.25 (1H, dd, J=6, 8 Hz), 8.12 (1H, bs), 7.2 (1H, bd, J=7.5Hz), 4.7 (2H, bt, 7 Hz), 3.9 (1H, m), 3.6 (3H, s), 2.0 (2H, m), 1.35(9H, s).

EXAMPLE 10 Preparation of 3-Quinolinemethanol

To a solution of 3.14 g (20 mmol) of 3-quinolinecarboxaldehyde in 60 mLof methanol was added portionwise 0.95 g (25 mmol) of sodium borohydrideat 0° C. The reaction mixture was then stirred at room temperature for 2hours. Excess sodium borohydride was quenched with acetic acid and thereaction mixture was evaporated in vacuo. The residue was dissolved indilute aqueous sodium bicarbonate and extracted three times with ethylacetate. The organic layer was washed with saturated brine, dried overanhydrous magnesium sulfate and evaporated in vacuo to give an oil. Theoil was chromatographed on silica gel, using first 3:2 chloroform/ethylacetate and then ethyl acetate as eluents, to afford 2.35 g (79.5%) ofthe title compound of the structural formula ##STR82## The product wascharacterized as follows: TLC R_(f) =0.30 (ethyl acetate); IR (neat) ν3120, 1580, 1500, 1060 cm⁻¹ ; NMR (CDCl₃) δ 8.73 (1H, d, J=2 Hz), 8.07(1H, s), 8.02 (1H, d, J=7 Hz), 7.8-7.3 (3H, m), 4.82 (2H, s).

EXAMPLE 11 Preparation of 3-Chloromethylquinoline hydrochloride

To a solution of 2.53 g (15.9 mmol) of 3-quinolinemethanol in 25 mL oftoluene was added dropwise at room temperature 5 mL of thionyl chloride.The reaction mixture was stirred at that temperature for 4 hours, thendried in vacuo to give which was suspended in 20 mL of toluene andtreated with 4 mL of thionyl chloride at room temperature overnight. Thereaction mixture was concentrated under reduced pressure to give 2.05 g(60%) of a solid. The product, having the structural formula ##STR83##was not further purified. NMR (DMSO-d₆) δ 9.36 (1H, d), 9.1 (1H, bs),8.6-7.8 (4H, m), 5.2 (2H, s).

EXAMPLE 12 Preparation of Diethyl (3-quinolylmethyl)acetamidomalonate

To a solution of sodium ethoxide [prepared from 0.25 g (11 mg-atoms) ofsodium metal] in 24 mL of ethanol was added, in one portion, 2.39 g (11mmol) of diethyl acetamidomalonate at room temperature. The reactionmixture was stirred for an additional 30 minute period. To that reactionmixture was then added a solution of 3-chloromethylquinoline [preparedfrom 2.05 g (9.25 mmol) of its hydrochloride salt by treatment withaqueous sodium bicarbonate and extraction with methylene chloride] in 5mL of ethanol. The resultant reaction mixture was refluxed overnight,then was evaporated in vacuo, dissolved in 1N hydrochloric acid andextracted with ethyl acetate. The aqueous layer was neutralized withsolid sodium bicarbonate and extracted twice with ethyl ether. The etherlayer was washed with saturated brine, dried over anhydrous magnesiumsulfate and evaporated in vacuo. The oil thus obtained waschromatographed on silica gel, using 2:1 chloroform/ethyl acetate aseluent, to afford a white crystalline product. Recrystallization from amixture of ethyl acetate and isopropyl ether gave 1.28 g (38.8%) of thepure compound of the formula ##STR84## melting at 124°-125° C.; thinlayer chromatography (R_(f) =0.28; chloroform/ethyl acetate, 2:1) and IRand NMR spectra confirmed the identity of the title compound.

EXAMPLE 13 Preparation of 3-(3-Quinolyl)alanine

A suspension of 1.19 g (3.34 mmol) of diethyl(3-quinolylmethyl)acetamidomalonate in 30 mL of 10% aqueous hydrochloricacid was refluxed overnight. The reaction mixture was then cooled toroom temperature and neutralized with 10% aqueous sodium hydroxidesolution. The precipitate was removed by filtration, washed with waterand dried in vacuo to yield 0.48 g (66.4%) of the desired product as awhite solid melting at 241°-243° C. Elemental analysis and IR confirmedthe identity of the product, which has the formula ##STR85##

EXAMPLE 14 Preparation of 1-3-(Benzyloxycarbonylamino)alanine

A solution of 5 g (35.5 mmol) of 2,3-diaminopropionic acid in 88.6 mL of0.05M phosphate buffer solution was cooled in ice. The pH of thesolution was adjusted to 6.7 with 10% aqueous sodium hydroxide solution.A solution of 7.6 mL (53.7 mmol) of benzyl chloroformate in 7.1 mL oftoluene was added dropwise, with vigorous stirring, over a one hourperiod. The pH was monitored and adjusted to between 6.5 and 7.0 by theaddition of 10% aqueous sodium hydroxide solution. The reaction mixturewas stirred with cooling at a pH of about 6.5 for an additional 5 hours,then was refrigerated overnight. The resulting precipitate was collectedby filtration and the solid was washed with water and ethyl ether.Crystallization from water gave 1.12 g (12.4%) of white crystals meltingat 233°-235° C. (with decomposition). The product has the structuralformula: ##STR86## and was subsequently prepared in 55.7% yield usingthe same general procedure.

EXAMPLE 15 Preparation of1-3-Benzyloxycarbonylamino-N-(tert-butoxycarbonyl)alanine

To a solution of 0.7 g (2.96 mmol) of1-3-(benzyloxycarbonylamino)alanine and 0.62 mL (4.4 mmol) oftriethylamine in 14 mL of 50% aqueous dioxane was added, in one portion,0.79 g (3.26 mmol) of2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile. Stirring wascontinued overnight at room temperature. The reaction mixture wasdiluted wit 14 mL of water and extracted twice with ethyl acetate. Theaqueous layer was acidified with 1N hydrochloric acid and extractedthree times with ethyl acetate. That organic layer was washed twice withwater, then once with saturated brine, dried over anhydrous magnesiumsulfate and evaporated in vacuo. The residue was chromatographed onsilica gel (15 g), using first chloroform and then ethyl acetate aseluents, to give 0.88 g (92.8%) of a colorless oil. NMR (CDCl₃) δ 8.0(1H, bs), 7.3 (5H, s), 6.2 (1H, m), 5.7 (1H, m), 5.1 (2H, bs), 4.3 (1H,m), 3.55 (2H, m), 1.4 (9H, s). The product has the structural formula:##STR87##

EXAMPLE 16 Preparation of1-3-Benzyloxycarbonylamino-N-(tert-butoxycarbonyl)alanylglycine ethylester

To a solution of 0.14 g (1 mmol) of glycine ethyl ester hydrochlorideand 0.14 mL (1 mmol) of triethylamine in 5 mL of chloroform was added asolution of 0.32 g (1 mmol) of1-3-benzyloxycarbonylamino-N-(tert-butoxycarbonyl)alanine in 3 mL ofchloroform. Then, 0.21 g (1 mmol) of dicyclohexylcarbodiimide was addedat room temperature and the reaction mixture was stirred at thattemperature overnight. The reaction mixture was diluted with chloroform,washed successively with 0.5N hydrochloric acid, water, 3% aqueoussodium bicarbonate solution and water, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waschromatographed on silica gel (10 g), using first 5:1 chloroform/ethylacetate and the 1:1 chloroform/ethyl acetate as eluents, to give a whitesolid. Crystallization from a mixture of ethyl acetate and isopropylether afforded 0.25 g (60.8%) of the title compound melting at 131°-132°C. and having the structural formula: ##STR88## The structure wasconfirmed by NMR analysis.

EXAMPLE 17 Preparation ofl-1-[2-tert-Butoxycarbonylamino-2-(N-ethoxycarbonylmethyl)carbamoyl]ethyl-3-carbamoylpyridiniumchloride

A mixture of 0.192 g (0.467 mmol) of1-3-benzyloxycarbonylamino-N-(tert-butoxycarbonyl)alanylglycine ethylester, 60 mg of 10% palladium on carbon catalyst and 6 mL of methanolwas vigorously stirred for 3 hours at room temperature under a hydrogenatmosphere. The catalyst was removed by filtration and the filtrate wasmixed with 0.15 g (0.467 mmol) of3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride. The resultantreaction mixture was refluxed overnight. The precipitate was thenremoved by filtration and the filtrate was evaporated in vacuo. Theresidue was chromatographed on silica gel using first acetonitrile andthen 10% aqueous acetonitrile as eluents, to give 20 mg of a brown oil.NMR (DMSO-d₆) δ 9.9 (1H, bs), 9.6 (1H, m), 9.3 (2H, m), 9.0 (1H, m),8.35 (1H, q), 8.2 (1H, bs), 7.3 (1H, bd), 6.0 and 5.3 (1H, m), 4.8 (2H,m), 4.13 (2H, q), 3.95 (2H, m), 1.2 (12H, m). The product has thestructural formula: ##STR89##

Preparation of 3-(3-Quinolyl)alanine ethyl ester dihydrochloride

A mixture of 0.45 g (2.08 mmol) of 3-(3-quinolyl)-alanine, 1 mL ofthionyl chloride and 20 mL of ethanol was gently refluxed overnight. Thereaction mixture was concentrated under reduced pressure. The residuewas dissolved in water and treated with active charcoal. Removal of theactive charcoal by filtration afforded a filtrate which was evaporatedto give a white solid. Crystallization from a mixture of ethanol andethyl ether gave 0.598 g (90.6%) of the title compound, melting at204°-205° C. with decomposition. NMR spectrum was consistent with theassigned structure: ##STR90##

EXAMPLE 19 Preparation of N-(tert-Butoxycarbonyl)-3-(3-quinolyl)-alanineethyl ester

A mixture of 0.58 g (1.84 mmol) of 3-(3-quinolyl)-alanine ethyl esterdihydrochloride, 0.78 mL (5.4 mmol) of triethylamine, 0.49 g (2.03 mmol)of 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile and 12 mL of 50%aqueous dioxane was stirred overnight at room temperature. The reactionmixture was then diluted with 15 mL of water and extracted three timeswith ethyl acetate. The organic layer was washed, first with water andthen with saturated brine, then was dried over anhydrous magnesiumsulfate and evaporated in vacuo. The residue was chromatographed onsilica gel, using first 5:1 chloroform/ethyl acetate and then 2:1chloroform/ethyl acetate as eluents, to give 0.486 g (76.7%) of acolorless oil. TLC R_(f) =0.4 (chloroform/ethyl acetate, 2:1); NMR(CDCl₃) δ 8.7 (1H, d), 8.1 (1H, bd), 7.93 (1H, d), 7.9-7.4 (3H, m), 5.15(1H, m), 4.63 (1 H, m), 4.2 (2H, m), 3.3 (2H, m), 1.4 (9H, s), 1.3 (3H,t). The product has the structural formula: ##STR91##

EXAMPLE 20 Preparation of3-[2-(tert-Butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide

A mixture of 0.486 g (1.41 mmol) of(N-tert-butoxycarbonyl)-3-(3-quinolyl)alanine ethyl ester, 2 mL ofmethyl iodide and 3 mL of acetone was refluxed overnight. The reactionmixture was evaporated in vacuo and the residue was chromatographed onsilica gel, using first acetonitrile and then 2% aqueous acetonitrile aseluents, to give a yellow solid. Crystallization from a mixture ofacetone and ethyl acetate yielded 0.497 g (72.5%) of a crystallineproduct melting at 98°-102° C. NMR spectrum and elemental analysis wereconsistent with the assigned structure: ##STR92##

EXAMPLE 21 Preparation ofN-(tert-Butoxycarbonyl)-3-(1,2-dihydro-1-methylquinolin-3-yl)alanineethyl ester

To a solution of 0.2 g (0.41 mmol) of3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide in 10 mL of ethanol was added dropwise a solution of 20 mg (0.5mmol) of sodium borohydride in 1 mL of ethanol at 0° C. After 5 minutes,the solution became colorless. The reaction mixture was diluted with 40mL of deaerated water and extracted twice with deaerated ethyl ether.The combined organic layers were washed, first with deaerated water andthen with saturated brine, then were dried over anhydrous magnesiumsulfate and evaporated in vacuo to give 0.146 g (99.1%) of a yellow oil.TLC R_(f) =0.8 (4% methanol in chloroform); NMR (CDCl₃) δ 7.2-6.4 (4H,m), 6.15 (1H, bs), 5.05 (1H, bd), 4.4 (1H, m), 4.2 (2H, q), 3.9 (2H,bs), 2.75 (3H, s), 2.55 (2H, m), 1.4 (9H, s), 1.25 (3H, t). The majorproduct has been assigned the structural formula: ##STR93## Analternative to the title nomenclature for this product is3-[2-tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1,2-dihydro-1-methylquinoline.

EXAMPLE 22 Preparation ofN-(tert-Butoxycarbonyl)-3-(1,4-dihydro-1-methylquinolin-3-yl)alanineethyl ester

To a solution of 0.2 g (0.41 mmol) of3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide in 20 mL of deaerated water were added 0.2 g (2.4 mmol) of sodiumbicarbonate and 20 mL of deaerated ethyl ether. The reaction mixture wasstirred in an ice water bath and 0.29 g (1.64 mmol) of sodium dithionitewas added in one portion. The reaction mixture was stirred for anadditional 2 hour period at 0° C. The organic layer was separated,washed first with deaerated water and then with saturated brine, driedover anhydrous magnesium sulfate and evaporated in vacuo to give 76.6 mg(51.8%) of a yellow oil. TLC R_(f) =0.77 (4% methanol in chloroform);NMR (CDCl₃) δ 7.2-6.5 (4H, m), 5.8 (1H, m), 5.0 (1H, m), 4.35 (1H, m),4.2 (2H, q), 3.5 (2H, bs), 3.0 (3H, s), 2.4 (2H, m), 1.49 (9H, s), 1.25(3H, t). The major product has been assigned the structure: ##STR94## Analternative to the title nomenclature for this product is3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1,4-dihydro-1-methylquinoline.

EXAMPLE 23 Preparation of Diethyl β-(p-methoxyanilino)methylenemalonate

A mixture of 24.6 g (0.2 mol) of p-anisidine and 43.2 g (0.2 mol) ofdiethyl ethoxymethylenemalonate was heated at 130° C. until no morebubbles of alcohol could be detected. The reaction mixture was cooled ina dry ice/methanol bath, affording a solid mass. To that residue wereadded 40 mL of ethyl ether and the dark brown solid was crushed wellwith the ether to form white crystals. The solid was collected on apreviously cooled Buchner funnel to give 42.48 g (72.4%) of a gray solidmelting at 38°-39° C. IR (KBr) ν 1680, 1640, 1620, 1595, 1525, 1230 cm⁻¹; NMR (acetone-d₆) δ 8.4 (1H, d), 7.3 (2H, d), 6.95 (2H, d), 4.2 (4H,m), 3.8 (3H, s), 1.3 (6H, dt). The product has the structural formula:##STR95##

EXAMPLE 24 Preparation of1,4-Dihydro-3-ethoxycarbonyl-6-methoxy-4-oxoquinoline

To 200 mL of boiling diphenyl ether were added rapidly 42 g (0.14 mol)of diethyl β-(p-methoxyanilino)methylenemalonate. The dark solution washeated under reflux for 30 minutes, then was cooled to room temperatureand diluted with 200 mL of n-hexane. The brown solid which formed wascollected, washed twice with n-hexane and then twice with ethyl etherand then dried in vacuo. The resultant solid was suspended in 60 mL ofethanol and the suspension was boiled for 30 minutes, then cooled toroom temperature. The product was collected as a pale brown solid (19.34g, 52.1%), melting at 274°-277° C. IR (KBr) ν 3600-2600, 1685, 1620,1575, 1560, 1520, 1480, 1290, 1170 cm⁻¹ ; NMR (CDCl₃) δ 8.45 (1H, s),7.6 (2H, two d), 7.3 (1H, dd), 4.2 (2H, q), 3.8 (3H, s), 1.3 (3H, t).The compound is further characterized by the structural formula##STR96##

EXAMPLE 25 Preparation ofL-1-[5-(tert-Butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoyl-1,4-dihydropyridine

To a solution of 0.2 g (0.5 mmol) ofL-1-[5-(tert-butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoylpyridiniumchloride in 20 mL of deaerated water were added 0.25 g (3 mmol) ofsodium bicarbonate and 20 mL of deaerated ethyl ether. The reactionmixture was cooled, with stirring, in an ice bath and 0.35 g of sodiumdithionite was added in one portion. The reaction mixture was stirredfor an additional one hour at 0° C. The organic layer was separated,washed with deaerated water and saturated brine, dried over anhydrousmagnesium sulfate and evaporated in vacuo to give 41 mg (23.3%) of ayellow oil. TLC R_(f) =0.57 (chloroform/methanol), 5:1); NMR (CDCl₃) δ7.0 (1H, d); 5.7 (1H, m), 5.5 (1H, m), 5.2 (1H, bd), 4.7 (1H, dt), 4.3(1H, m), 3.75 (3H, s), 3.15 (2H, m), 1.49 (9H, s). The product has thestructural formula: ##STR97##

EXAMPLE 26 Preparation ofL-1-[5-tert-Butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoyl-1,4-dihydropyridine

To a solution of 0.2 g (0.5 mmol) ofL-1-[5-tert-butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoylpyridiniumchloride in 20 mL of deaerated methanol was added 20 mg of sodiumborohydride at 0° C. The reaction mixture was stirred for an additional30 minutes at that temperature, then was evaporated in vacuo an dilutedwith deaerated water. Extraction with deaerated ethyl ether afforded anorganic layer, which was washed with deaerated water and saturatedbrine, dried over anhydrous magnesium sulfate and evaporated in vacuo.Obtained in this manner was a complex mixture which was shown by NMRspectral analysis to contain the 1,4-dihydro derivative and the1,6-dihydro derivative. The residue was chromatographed on silica gel(packed by 5% triethylamine in methylene chloride and eluted with 1:9ethyl acetate/chloroform containing 0.5% triethylamine) to give 21 mg(12%) of the title 1,4-dihydro derivative as a yellow oil, identical tothe product of EXAMPLE 25.

EXAMPLE 27 Preparation of 4-Chloro-3-ethoxycarbonyl-6-methoxyquinoline

A mixture of 19 g (76.8 mmol) of1,4-dihydro-3-ethoxycarbonyl-6-methoxy-4-oxoquinoline and 60 mL ofphosphorus oxychloride (POCl₃) was refluxed for 4 hours. Excessphosphorus oxychloride was evaporated and the residue was poured onto100 g of ice, made alkaline with 28% ammonium hydroxide, and allowed toremain at room temperature. The precipitate which formed was collected,washed twice with water and dried in vacuo to give 20 g of the productas a pale brown solid, in quantitative yield. TLC R_(f) =0.41 (2%methanol in chloroform); NMR (CDCl₃) δ 8.95 (1H, s), 8.05 (1H, bd), 7.6(2H, m), 4.45 (2H, q), 4.0 (3H, s), 1.4 (3H, t); IR (KBr) ν 1740, 1620,1580, 1500, 1295, 1230, 1020, 830 cm⁻¹. The product has the formula:##STR98##

EXAMPLE 28 Preparation of 3-Ethoxycarbonyl-6-methoxyquinoline

A mixture of 20 g (75.8 mmol) of3-ethoxycarbonyl-4-chloro-6-methoxyquinoline, 1.5 g of 10% palladium oncarbon catalyst, 20 mL of triethylamine and 300 mL of ethanol was shakenunder a hydrogen atmosphere (30 psi) at room temperature overnight. Thecatalyst was removed by filtration and the filtrate was evaporated. Theresidue was dissolved in methylene chloride, washed with aqueous sodiumbicarbonate solution and water, dried over anhydrous sodium sulfate andevaporated in vacuo. That residue was suspended in 500 mL of cyclohexaneand the resultant suspension was refluxed, then filtered to removeinsoluble materials. The filtrate was allowed to stand at roomtemperature and the crystalline material which formed was collected togive 9.18 g (51.5%) of a pale brown solid having the structural formula:##STR99## NMR (CDCl₃) δ 9.25 (1H, d), 8.7 (1H, d), 8.05 (1H, d, J=9 Hz),7.5 (1H, dd, J=9 Hz, 3 Hz), 7.2 (1H, d, J=3 Hz), 4.45 (2H, q), 3.9 (3H,s), 1.45 (3H, t). IR (KBr) ν 1713, 1620, 1600, 1500, 1240, 1100, 1025cm⁻¹.

EXAMPLE 29 Preparation of 3-Hydroxymethyl-6-methoxyquinoline

To a suspension of 5.9 g (25 mmol) of3-ethoxycarbonyl-6-methoxyquinoline in 120 mL of 1:1 toluene/methylenechloride were added dropwise at -50° C., 36.7 mL (55 mmol) ofdiisobutylaluminium hydride as a 1.5M solution in toluene. The reactionmixture was gradually warmed to room temperature. To this mixture wereadded about 4 mL of methanol to quench excess reducing agent, and thenwater was added with vigorous stirring. After 30 minutes, 150 mL ofethyl acetate and anhydrous magnesium sulfate were added and the mixturewas stirred for an additional 30 minutes. The solid was removed byfiltration and washed thoroughly with ethyl acetate. The filtrate andwashings were combined and evaporated in vacuo. The residue waschromatographed on silica gel, using first ethyl acetate and then 2%ethanol in ethyl acetate as eluents, to give 1.72 g (36.4%) of the titlecompound as a solid which melts at 92°-93° C. after crystallization froma mixture of benzene and isopropyl ether. TLC R_(f) =0.17 (ethylacetate); NMR (CDCl₃) δ 8.6 (1H, d), 7.9 (2H, m), 7.3 (1H, q), 6.95 (1H,d), 4.8 (2H, s), 3.85 (3H, s); IR (KBr) ν 3070, 1625, 1500, 1215 cm⁻¹.

A subsequent preparation using the same general procedure afforded thetitle compound in 71.5% yield.

EXAMPLE 30 Preparation of 3-Chloromethyl-6-methoxyquinolinehydrochloride

A mixture of 1.6 g (8.45 mmol) of 3-hydroxymethyl-6-methoxyquinoline, 3mL of thionyl chloride and 16 mL of toluene was refluxed overnight. Thereaction mixture was diluted with 16 mL of toluene. The solid wasseparated by filtration, washed thoroughly with toluene and dried invacuo to give 1.52 g (73.7%) of a pale brown solid: NMR (DMSO-d₆) δ 9.15(1H, d), 8.9 (1H, bs), 8.3 (1H, d), 7.7 (2H, m), 5.1 (2H, s), 3.95 (3H,s); IR (KBr) ν 2540, 2340, 2020, 1620, 1573, 1495, 1250 cm⁻¹. Theproduct has the structural formula: ##STR100##

A subsequent preparation using the same general procedure afforded thedesired compound in 87.0% yield.

EXAMPLE 31 Preparation of Diethyl(6-methoxy-3-quinolyl)methylacetamidomalonate

To a solution of sodium ethoxide prepared from 0.3 g (13.1 mmol) ofsodium and 30 mL of ethanol were added, in one portion, 2.85 g (13.1mmol) of diethyl acetamidomalonate. After 30 minutes, 1.52 g (6.24 mmol)of 3-chloromethyl-6-methoxyquinoline hydrochloride were added and theresultant mixture was refluxed overnight. The reaction mixture wasevaporated and the residue was dissolved in 25 mL of 1N hydrochloricacid and extracted twice with ethyl ether. The aqueous layer wasneutralized with solid sodium bicarbonate and extracted twice withchloroform. The chloroform layer was washed with water, dried overanhydrous magnesium sulfate and evaporated in vacuo. The residue waschromatographed on silica gel, using first 4:1 methylene chloride/ethylacetate and the 1:1 methylene chloride/ethyl acetate as eluents, to give1.82 g (75.2%) of the desired product as a white solid, which melted at151°-152° C. after crystallization from a mixture of ethyl acetate andisopropyl ether. TLC R_(f) =0.34 (ethyl acetate); NMR (CDCl₃) δ 8.4 (1H,d), 7.9 (1H, d), 7.7 (1H, bs), 7.35 (1H, d), 7.0 (1H, d), 6.6 (1H, bs),4.3 (4H, q), 3.9 (3H, s), 3.85 (2H, s), 2.05 (3H, s), 1.3 (6H, t); IR(KBr) ν 3400, 3230, 1740, 1640, 1300 cm⁻¹. The product has the formula:##STR101##

An improved yield of 89.3% was obtained when the above procedure wasrepeated subsequently.

EXAMPLE 32 Preparation of N.sup.ε -Benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysylglycine ethyl ester

To a solution of 0.73 g (5.26 mmol) of glycine ethyl ester hydrochlorideand 0.74 mL (5.26 mmol) of triethylamine in 42 mL of chloroform wereadded successively, at 0° C., 2.0 g (5.26 mmol) of N.sup.ε-benzyloxycarbonyl-N.sup.α -(tert-butoxycarbonyl)-L-lysine and 1.09 g(5.26 mmol) of dicyclohexylcarbodiimide. The reaction mixture wasstirred overnight at room temperature. The resulting precipitate wasremoved by filtration and the filtrate was washed successively with 0.5Nhydrochloric acid, water, 3% aqueous sodium bicarbonate solution andwater, then dried over anhydrous sodium sulfate. Evaporation of thesolvent afforded a residue which was triturated with ethyl acetate.Insoluble materials were removed by filtration and the filtrate wasevaporated in vacuo. The residue was recrystallized from a mixture ofethyl acetate and isopropyl ether to give 1.15 g (48%) of the titlecompound as colorless crystals. The mother liquor was concentrated underreduced pressure and the residue was crystallized as before to give asecond crop of crystals (0.77 g, 32.1%). Total yield 80.1%, meltingpoint 69°-70° C. NMR and IR spectral analyses confirmed the identity ofthe product, which has the formula: ##STR102##

EXAMPLE 33 Preparation of(S)-1-[5-(tert-Butoxycarbonyl)amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoylpyridiniumchloride

A mixture of 0.911 g (2 mmol) of N.sup.ε -benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysylglycine ethyl ester and 0.25 g of 10%palladium on carbon catalyst in 5 mL of ethanol was vigorously stirredfor 3 hours at room temperature under a hydrogen atmosphere. Thereaction mixture was then filtered through Celite® (a diatomaceous earthfilter aid) and to the filtrate was added, in one portion, 0.65 g (2mmol) of 3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride. Thereaction mixture was stirred at room temperature overnight, during whichtime the color changed from dark red to reddish brown. The greenishbrown precipitate which formed was removed by filtration and thefiltrate was evaporated in vacuo. That residue was chromatographed onsilica gel (using acetonitrile, 2% acetonitrile and 10% acetonitrilesuccessively as the eluents) to give a brown oil. The product was takenup in anhydrous acetonitrile, filtered through Celite®, evaporated anddried in vacuo to afford 0.603 g (65.1%) of the desired compound as ayellow solid. NMR (DMSO-d₆ ) δ 9.8 (1H, bs), 9.3 (1H, bd), 9.05 (1H,bd), 9.0 (1H, m), 8.45 (1H, t), 8.25 (1H, dd), 8.15 (1H, d), 6.8 (1H,m), 4.7 (2H, m), 4.05 (2H, q), 4.0 (1H, m), 3.8 (2H, m), 1.35 (9H, s),1.15 (3H, t). Elemental analysis was consistent with the assignedstructure: ##STR103## EXAMPLE 34

Preparation of(S)-1-[5-(tert-Butoxycarbonyl)amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoyl-1,4-dihydropyridine

To a mixture of 0.236 g (0.5 mmol) of(S)-1-[5-(tert-butoxycarbonyl)amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoylpyridiniumchloride, 0.25 g (3 mmol) of sodium bicarbonate, 11 mL of methylenechloride and 11 mL of water was added, in one portion at roomtemperature, 0.31 g (2.0 mmol) of sodium dithionite. The reactionmixture was stirred for an additional one hour period. The organic layerwas separated, dried over anhydrous sodium sulfate and evaporated invacuo to afford 0.19 g (87.1%) of a yellow oil. The product has thestructural formula ##STR104## NMR (acetone-d₆) δ (1H, m), 7.0 (1H, bs),6.3 (2H, bs), 6.2 (1H, d), 5.85 (1H, d), 4.65 (1H, dt), 4.15 (3H, q+m),4.0 (2H, m), 3.15 (4H, m), 1.4 (9H, s), 1.13 (3H, t). Elemental analysiswas in agreement with the assigned structure.

EXAMPLE 35 Preparation of Diethyl(6-hydroxy-3-quinolyl)methylacetamidomalonate

To a solution of 0.39 g (1 mmol) of diethyl(6-methoxy-3-quinolyl)methylacetamidomalonate in 10 mL of dry methylenechloride was added dropwise at -70° C. a 1 M solution of borontribromide in methylene chloride. The reaction temperature was graduallyraised to room temperature while stirring overnight. Then, the reactionmixture was poured into ice water and extracted with methylene chloride.The organic layer was washed with water, dried over anhydrous magnesiumsulfate and evaporated in vacuo. The residue was chromatographed onsilica gel, using 1:1 ethyl acetate/methylene chloride and then ethylacetate as eluents, to give 43 mg (11.5%) of the desired product as asolid. TLC R_(f) =0.46 (ethyl acetate); NMR (CDCl₃) δ 8.4 (1H, bs), 7.95(1H, d), 7.7 (1H, bs), 7.35 (1H, dd), 7.1 (1H, d), 6.8 (1H, bs), 4.25(4H, m), 3.8 (2H, bs), 2.0 (3H, s), 1.3 (6H, m); IR (KBr) ν 3700-2800,1740, 1660, 1645, 1620, 1500 cm⁻¹. The product has the structuralformula: ##STR105##

EXAMPLE 36 Preparation of 3-(6-Methoxy-3-quinolyl)alanine

A mixture of 0.602 g (1.55 mmol) of diethyl(6-methoxy-3-quinolyl)methylacetamidomalonate and 8.2 mL of 10%hydrochloric acid was refluxed overnight. The reaction mixture wascooled to room temperature and neutralized with 1N sodium hydroxidesolution to pH 6.7. The white solid which formed was separated byfiltration, washed successively with water and ethyl ether and dried invacuo to give 0.282 g (73.9%) of the title compound melting at 246°-247°C. and having the structural formula ##STR106## NMR and IR spectralanalyses and elemental analysis confirmed the identity of the product.

EXAMPLE 37 Preparation of 3-(6-Hydroxy-3-quinolyl)alanine

A mixture of 0.18 g (0.74 mmol) of 3-(6-methoxy-3-quinolyl)alanine and5.4 mL of 48% hydrobromic acid was stirred at 85°-90° C. overnight. Thereaction mixture was cooled to room temperature and neutralized to pH7.0 with 1N sodium hydroxide solution, then was concentrated underreduced pressure to give a brown solid. After filtration, the solidproduct was washed with a small amount of cold water and ethyl ether andthen was dried in vacuo to give 57.4 mg (33.4%) of the title compound ofthe formula: ##STR107## NMR (DMSO-d₆, CF₃ COOH) δ 9.05 (1H, bs), 8.9(1H, bs), 8.2 (1H, d), 7.7 (1H, dd), 7.5 (1H, d), 4.5 (1H, m), 3.5 (2H,m); IR (KBr) ν 3400, 1630, 1500 cm⁻¹.

EXAMPLE 38 Preparation ofN-(tert-Butoxycarbonyl)-3-(6-hydroxy-3-quinolyl)alanine ethyl ester

A mixture of 0.388 g (1 mmol) of diethyl(6-methoxy-3-quinolyl)methylacetamidomalonate and 7.4 mL of 48%hydrobromic acid was stirred at 85° C. for 24 hours. The solvent wasevaporated and the residue was dissolved in 7 mL of ethanol. Then, 0.3mL of thionyl chloride was added at 0° C. and the mixture was refluxedovernight. The reaction mixture was evaporated in vacuo, taken up inbenzene and the evaporated to dryness in vacuo to give a crude, highlyhygroscopic residue. To a mixture of this crude product together with0.39 mL (2.7 mmol) of triethylamine and 6 mL of 50% aqueous 1,4-dioxanethere was added, in one portion, 0.24 g (1 mmol) of2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile. The reactionmixture was stirred overnight at room temperature, then was diluted with6 mL of water and extracted twice with chloroform. The organic layer waswashed with water, dried over anhydrous magnesium sulfate and evaporatedin vacuo. The residue was chromatographed on silica gel. Elution with5:1 methylene chloride/ethyl acetate gave 94 mg (27.6%) of the methoxyderivative, i.e. N-(tert-butoxycarbonyl)-3-(6-methoxy-3-quinolyl)alanineethyl ester, having the formula: ##STR108## TLC R_(f) =0.57 (1:1methylene chloride/ethyl acetate); NMR (CDCl₃) δ 8.55 (1H, d), 8.0 (1H,d), 7.85 (1H, bs), 7.35 (1H, dd), 7.0 (1H, d), 5.1 (1H, m), 4.65 (1H,m), 4.2 (2H, q), 3.9 (3H, s), 3.3 (2H, m), 1.4 (9H, s), 1.2 (3H, t).

The title compound was eluted with 1:1 methylene chloride/ethyl acetateto give 0.14 g or 42.7% yield; TLC R_(f) =0.31 (1:1 methylenechloride/ethyl acetate); melting point 176°-177° C. aftercrystallization from a mixture of ethyl acetate and isopropyl ether. Thetitle compound has the formula ##STR109## NMR (CDCl₃) δ 8.55 (1H, d),7.95 (1H, d), 7.8 (1H, d), 7.35 (1H, dd), 7.15 (1H, d), 5.3 (1H, m), 4.6(1H, m), 4.15 (2H, q), 3.25 (2H, m), 1.35 (9H, s), 1.2 (3H, t); IR (KBr)ν 3350, 1740, 1710, 1685, 1630, 1500, 1160 cm⁻¹.

EXAMPLE 39 Preparation ofL-1-[2-(N-tert-Butoxycarbonyl)amino-2-(N-ethoxycarbonylmethyl)carbamoyl]ethyl-3-carbamoylpyridiniumchloride

A mixture of 0.81 g (1.92 mmol) of3-benzyloxycarbonylamino-N-(tert-butoxycarbonyl)-L-alanylglycine ethylester, 0.2 g of 10% palladium on carbon catalyst and 30 mL of ethanolwas stirred vigorously at room temperature for 4 hours under a hydrogenatmosphere. The reaction mixture was filtered and the solid residue waswashed with 10 mL of ethanol. The filtrate and washing were combined and0.623 g (1.92 mmol) of 3-carbamoyl-1-(2,4-dinitrophenyl)pyridiniumchloride was added in one portion. Stirring was continued at roomtemperature overnight, then the reaction mixture was filtered and thefiltrate was evaporated in vacuo. The residue was chromatographed onsilica gel, using first 2% aqueous acetonitrile and then 5% aqueousacetonitrile as eluents. The eluate was concentrated to approximately100 mL under reduced pressure and the was diluted with 300 mL withwater. The resulting solution was concentrated to approximately 100 mLand treated with active charcoal. Removal of the solid by filtrationgave a filtrate which was lyophilized to give 0.202 g (24.4%) of thetitle compound as a white solid. That compound, which can alternately beprepared by the process of EXAMPLE 3, can also be named asN-(tert-butoxycarbonyl)-3-(3-carbamoyl-1-pyridinium)-L-alanylglycineethyl ester. It has the structure depicted in EXAMPLE 3.

EXAMPLE 40 Preparation of N.sup.ε -Benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysyl-L-phenylalanyl-L-leucine ethyl ester

To a mixture of 1.68 g (4 mmol) of L-phenylalanyl-L-leucine ethyl estertrifluoroacetate, 0.56 mL (4 mmol) of triethylamine and 32 mL ofchloroform were added at 0° C., first 1.52 g (4 mmol) of N.sup.ε-benzyloxycarbonyl-N.sup.α -(tert-butoxycarbonyl)-L-lysine, and the 0.83g (4 mmol) of dicyclohexylcarbodiimide, each being added in a singleportion. The reaction mixture was warmed gradually to room temperatureand then stirred overnight at that temperature. The precipitate whichformed was removed by filtration. The filtrate was evaporated in vacuoand the residue was chromatographed on silica gel using 3:1 methylenechloride/ethyl acetate as eluent to give 2.48 g (92.7%) of the titlecompound as a white solid melting at 135°-136° C. after crystallizationfrom a mixture of ethyl acetate and isopropyl ether. The product has thestructural formula: ##STR110## as further confirmed by NMR, IR andelemental analyses.

EXAMPLE 41 Preparation ofL-[2-(tert-Butoxycarbonyl)amino-6-(3-carbamoyl-1-pyridinium)]hexanoyl-L-phenylalanyl-L-leucineethyl ester chloride

A mixture of 1.0 g (1.5 mmol) of N.sup.ε -benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysyl-L-phenylalanyl-L-leucine ethyl ester,0.25 g of 10% palladium on carbon catalyst and 10 mL of ethanol wasstirred vigorously for 3 hours at room temperature under a hydrogenatmosphere. The reaction mixture was filtered and the residue was washedwith 5 mL of ethanol. The filtrate and washing were combined and 0.49 g(1.5 mmol) of 3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride wasadded in one portion at room temperature. The reaction mixture wasstirred overnight at room temperature and then filtered. The filtratewas evaporated in vacuo and the residue was chromatographed on silicagel using 2% aqueous acetonitrile and then 8% aqueous acetonitrile aseluents. The eluate was concentrated to approximately 100 mL underreduced pressure, then was diluted with 300 mL of water and againevaporated to approximately 100 mL in vacuo. The resulting solution wastreated with active charcoal and filtered. The filtrate was lyophilizedto give 0.501 g (49.4%) of a very hygroscopic solid. NMR (DMSO-d₆) δ9.65 (1H, bs), 9.2 (1H, bd), 9.0 (1H, bd), 8.8 (1H, bs), 8.5-8.1 (3H,m), 7.9 (1H, bd), 7.2 (5H, s), 6.85 (1H, m), 4.6 (4H, m), 4.1 (2H, q),1.35 (9H, s), 0.9 (6H, m). The title compound prepared in this mannerhas the structural formula: ##STR111##

EXAMPLE 42 Preparation of3-[2-(N-tert-Butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-6-hydroxy-1-methylquinoliniumiodide

A mixture of 0.34 g (0.94 mmol) ofN-(tert-butoxycarbonyl)-3-(6-hydroxy-3-quinolyl)alanine ethyl ester, 1.3mL of methyl iodide and 2 mL of acetone was gently refluxed overnight.Evaporation in vacuo afforded a residue which was recrystallized from amixture of acetone and ethyl acetate to give 0.39 g (82.6%) of the titlecompound as a yellow solid melting at 174°-175° C. NMR, IR and elementalanalyses were consistent with the assigned structure: ##STR112##

EXAMPLE 43 Preparation ofN-(tert-Butoxycarbonyl)-3-(6-methoxy-3-quinolyl)alanine ethyl ester

To a suspension of 0.54 g (2.19 mmol) of 3-(6-methoxy-3-quinolyl)analinein 13.5 mL of ethanol was added dropwise 0.68 mL of thionyl chloride at0° C. The reaction mixture was refluxed overnight, then was evaporatedin vacuo, triturated with benzene and evaporated to dryness in vacuo.The crude product, 3-(6-methoxy-3-quinolyl)alanine ethyl esterdihydrochloride, was combined with 0.93 mL (6.4 mmol) of triethylamineand 14.2 mL of 50% aqueous 1,4-dioxane; to that mixture was added, inone portion, 0.58 g (2.4 mmol) of2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile. The resultantmixture was stirred overnight at room temperature, then was diluted with15 mL of water and extracted twice with chloroform. The organic layerwas washed with water, dried over anhydrous magnesium sulfate andevaporated in vacuo. The residue was chromatographed on silica gel,using 5:1 methylene chloride/ethyl acetate as eluent, to give a whitesolid which was recrystallized from isopropyl ether to afford 0.6 g(73.3 %) of the title compound, melting at about 127° C. and having thestructural formula: ##STR113## The identifying of the product wasconfirmed by NMR and IR spectral analyses as well as by elementalanalysis.

EXAMPLE 44 Preparation of3-[2-N-(tert-Butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-6-methoxy-1-methyl-3-quinoliniumiodide

A mixture of 0.415 g (1.11 mmol) ofN-(tert-butoxycarbonyl)-3-(6-methoxy-3-quinolyl)alanine ethyl ester, 2mL of methyl iodide and 3 mL of acetone was gently refluxed overnight.Evaporation in vacuo afforded a residue which was chromatographed onsilica gel, using first 3:1 ethyl acetate/acetonitrile and then 1:2ethyl acetate/acetonitrile as eluents, to give 0.53 g (93.1%) of ayellow amorphous solid. The title compound was further characterized asfollows: NMR (DMSO-d₆) δ 9.37 (1H, bs), 8.9 (1H, bs), 8.4 (1H, d), 7.85(1H, dd), 7.8 (1H, s), 7.4 (1H, m), 4.6 (3H, s), 4.5 (1H, m), 4.12 (2H,q), 4.0 (3H, s), 3.3 (2H, m), 1.27 (9H, s), 1.17 (3H, t). Elementalanalysis further confirmed that the product was of the structuralformula: ##STR114##

EXAMPLE 45 Preparation ofN-(tert-Butoxycarbonyl)glycyl-3-(3-quinolyl)alanine ethyl ester

To a mixture of 0.637 g (2 mmol) of 3-(3-quinolyl)alanine ethyl esterdihydrochloride, 0.35 g (2 mmol) of N-(tert-butoxycarbonyl)glycine, 0.56mL (4 mmol) of triethylamine and 16 mL of chloroform was added at 0° C.,in one portion, 0.42 g (2 mmol) of dicyclohexylcarbodiimide. Thereaction mixture was stirred overnight and the precipitate which formedwas removed by filtration. The filtrate was washed twice with water,dried over anhydrous magnesium sulfate and evaporated in vacuo. Theresidue was chromatographed on silica gel, using first 1:1 methylenechloride/ethyl acetate and then 1:3 methylene chloride/ethyl acetate aseluents, to give 0.73 g (9.9%) of the title compound as a colorless oil.TLC R_(f) =0.52 (ethyl acetate); NMR (CDCl₃) δ 8.64 (1H, d), 7.9-8.1(2H, m), 7.9-7.4 (4H, m), 7.1 (1H, bd), 5.46 (1H, bt), 4.9 (1H, m), 4.15(2H, q), 3.8 (2H, bd), 3.3 (2H, m), 1.37 (9H, s) 1.2 (3H, t). Theproduct has the formula: ##STR115##

EXAMPLE 46 Preparation of3-{2-[N-(tert-Butoxycarbonyl)glycyl]amino-2-ethoxycarbonyl}ethyl-1-methylquinoliniumiodide

A mixture of 0.55 g (1.37 mmol) ofN-(tert-butoxycarbonyl)glycyl-3-(3-quinolyl)alanine ethyl ester, 1.9 mLof methyl iodide and 2.9 mL of acetone was gently refluxed overnight.The solvent was removed by evaporation and the residue waschromatographed on silica gel, using first 2:1 ethylacetate/acetonitrile and then 1:3 ethyl acetate/acetonitrile as eluents,to give 0.67 g (90.1%) of the title compound as a yellow amorphoussolid. NMR (DMSO-d₆) δ 9.5 (1H, s), 9.1 (1H, s), 8.6-7.9 (5H, m), 6.9(1H, m), 4.8 (1H, m), 4.63 (3H, s), 4.15 (2H, q), 3.45 (4H, m), 1.27(9H, s), 1.2 (2H, t). The product has the formula: ##STR116##

EXAMPLE 47 Preparation ofL-[2-(tert-Butoxycarbonyl)amino-6-(3-carbamoyl-1,4-dihydropyridin-1-yl)]hexanoyl-L-phenylalanyl-L-leucineethyl ester

To a mixture of 0.1 g (0.15 mmol) ofL-[2-(tert-butoxycarbonyl)amino-6-(3-carbamoyl-1-pyridinium)]hexanoyl-L-phenylalanyl-L-leucineethyl ester, 74 mg (0.89 mmol) of sodium bicarbonate, 5 mL of deaeratedmethylene chloride and 5 mL of deaerated water, there were added 92 mg(0.59 mmol) of sodium dithionite in one portion. The reaction mixturewas stirred at room temperature for 2 hours, then the organic layer wasseparated and washed with deaerated water. Drying over anhydrous sodiumsulfate and evaporation in vacuo afforded a brown amorphous product: NMR(CDCl₃) δ 7.25 (5H, s), 7.05 (1H, bs), 6.85 (1H, bd), 5.7 (3H, m), 5.3(1H, bd), 4.7 (2H, m), 4.5 (1H, m), 4.15 (2H, q), 4.1 (1H, m), 3.1 (4H,m), 2.5 (2H, m), 1.4 (9H, s), 1.25 (3H, t), 0.9 (6H, m). The identity ofthe title compound was confirmed by elemental analysis. It has thestructural formula: ##STR117##

EXAMPLE 48 Preparation ofO-Benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysine methyl ester

A solution of 1.04 g (2.64 mmol) of N.sup.ε -benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysine methyl ester in 5.3 mL oftrifluoroacetic acid was stirred for 2 hours at room temperature. Thereaction mixture was evaporated in vacuo to give a colorless oil, whichwas mixed with 0.966 g (2.6 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosine, 0.37 mL (2.6 mmol) oftriethylamine and 20.8 mL of chloroform. To that mixture was added, inone portion, 0.54 g (2.6 mL) of dicyclohexylcarbodiimide at 0° C. Thereaction mixture was stirred for one hour at 0° C. and then overnight atroom temperature. The solvent was removed by evaporation and the residuewas chromatographed on silica gel, using first methylene chloride andthen 1:1 methylene chloride/ethyl acetate as eluents, to give 0.49 g(29.1%) of a colorless solid. NMR (CDCl₃) δ 7.3 (10 H, m), 7.1 (2H, d),6.85 (2H, d), 6.5 (1H, m), 5.1 (2H, s), 5.0 (1H, m), 4.7-4.1 (2H, m),3.65 (3H, s), 3.3-2.9 (4H, m), 1.4 (9H, s). A portion of the product wasrecrystallized from a mixture of ethyl acetate and isopropyl ether togive crystals melting at 167°-168° C. The product has the formula##STR118## as confirmed by elemental analysis.

EXAMPLE 49 Preparation of1-{5-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-5-methoxycarbonyl}pentyl-3-carbamoylpyridiniumchloride

A mixture of 0.44 g (0.68 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysine methyl ester, 90 mg of 10% palladium oncarbon catalyst, 5 mL of methanol and 5 mL of dioxane was vigorouslystirred for 1 hour under a hydrogen atmosphere. The reaction mixture wasfiltered and to the filtrate was added 0.22 g (0.68 mmol) of3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride. That mixture wasstirred overnight at room temperature and then evaporated. The residuewas chromatographed on silica gel, using first acetonitrile and then 4%aqueous acetonitrile as eluents, to give a reddish brown oil, which wasdissolved in distilled water, treated with active charcoal andlyophilized to afford 0.106 g (27.7%) of a colorless powder; NMR (D₂ O,DSS) δ 9.65 (1H, bs), 9.3 (3H, m), 8.95 (2H, m), 8.5 (1H, dd), 8.25 (1H,d), 7.1 (2H, d), 6.8 (2H, d), 4.3 (2H, m), 3.7 (5H, m), 1.4 (9H, s). Theproduct has the formula: ##STR119##

EXAMPLE 50 Preparation ofO-Benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycine ethyl ester

A mixture of 0.94 (2 mmol) of N.sup.ε -benzyloxycarbonyl-N.sup.α-(tert-butoxycarbonyl)-L-lysylglycine ethyl ester and 4 mL oftrifluoroacetic acid was stirred for 40 minutes at room temperature. Thereaction mixture was diluted with 30 mL of toluene and evaporated toapproximately 3 mL. To this residue were added 40 mL of dry ethyl ester.Decantation of the solvent left a viscous oil which was mixed with 0.74g (2 mmol) of O-benzyl-N-(tert-butoxycarbonyl)-L-tyrosine, 0.28 mL (2mmol) of triethylamine and 16 mL of chloroform. Then, 0.42 g (2 mmol) ofdicyclohexylcarbodiimide was added in one portion and the reactionmixture was stirred overnight at room temperature. The precipitate whichformed was removed by filtration and the filtrate was washedsuccessively with 0.5% aqueous hydrochloric acid, water, 3% aqueoussodium bicarbonate solution and water, then dried over anhydrous sodiumsulfate. Evaporation of the solvent afforded a residue which waschromatographed on silica gel, using first 2:1 methylene chloride/ethylacetate, and then 1:1 methylene chloride/ethyl acetate as eluents, togive 1.17 g (81.4%) of a colorless solid. NMR (CDCl₃) δ 7.3 (10H, m),7.1 (2H, d), 6.85 (2H, d), 5.2 (1H, m), 5.1 (2H, s), 5.0 (2H, s), 4.4(2H, m), 4.15 (2H, q), 3.9 (2H, d), 3.1 (4H, m), 1.4 (9H, s), 1.2 (3H,t); IR (KBr) ν 3300, 1690, 1640, 1510, 1240, 1175, 1025 cm⁻¹. A portionof the product was recrystallized from a mixture of isopropyl ether andethyl acetate to afford a sample melting at 122°-124° C. Elementalanalysis gave values in accord with the following assigned structure:##STR120##

EXAMPLE 51 Preparation of1-{5-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-5-ethoxycarbonylmethylcarbamoyl}pentyl-3-carbamoylpyridiniumchloride

A mixture of 0.72 g (1 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycine ethyl ester, 0.15 g of 10% palladiumon carbon catalyst and 15 mL of ethanol was vigorously stirred for 2hours under a hydrogen atmosphere. The catalyst was removed byfiltration and 0.325 g (1 mmol) of3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride was added to thefiltrate. The reaction mixture was stirred overnight at roomtemperature. The solvent was removed by evaporation and the residue waschromatographed on silica gel, twice using acetonitrile, 3% aqueousacetonitrile and 6% aqueous acetonitrile successively as eluents, togive a reddish brown oil. The product was dissolved in distilled water,treated with active charcoal and lyophilized to give 0.109 g (17.1%) ofa colorless solid; NMR (CD₃ OD) δ 9.8 (1H, m), 9.4 (2H, m), 9.3 (1H, d),8.95 (1H, dd), 8.5 (1H, dd), 8.4 (1H, d), 8.0 (1H, m), 7.1 (2H, d), 6.7(2H, d), 4.15 (2H, q), 3.9 (2H, m), 3.7 (2H, m), 3.0 (4H, m), 1.4 (9H,s), 1.25 (3H, t). The product has the structural formula: ##STR121##

EXAMPLE 52 Preparation ofO-Benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycine

A suspension of 0.608 g (0.85 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycine ethyl ester in 60 mL of 50% aqueousmethanol was adjusted to pH 12.5 with 1N aqueous sodium hydroxidesolution and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was then diluted with 50 mL of waterand concentrated to about half of the original volume. The concentratedreaction mixture was adjusted to pH 3 with 1N aqueous hydrochloric acid,then was extracted twice with ethyl acetate. The organic layer waswashed with water and saturated brine, dried over anhydrous magnesiumsulfate and evaporated to give 0.56 g (93.5%) of a white solid. NMR (CD₃OD) δ 7.4 (10H, m), 7.2 (2H, d), 6.9 (2H, d), 5.05 (4H, s), 4.3 (2H, m),1.4 (9H, s); IR (KBr) ν 3500, 1670, 1640, 1510, 1240, 1160 cm⁻¹. Theproduct has the structural formula: ##STR122##

EXAMPLE 53 Preparation ofO-Benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycyl-L-phenylalanyl-L-leucine ethyl ester

To a mixture of 0.56 g (0.79 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycine, 0.40 g (0.95 mmol) ofL-phenylalanyl-L-leucine ethyl ester trifluoroacetate, 0.112 g (0.83mmol) of 1-hydroxybenztriazole hydrate, 0.13 mL (0.95 mmol) oftriethylamine and 8 mL of dimethylformamide was added, in one portion at0° C., 0.17 g (0.83 mmol) of dicyclohexylcarbodiimide. The reactionmixture was stirred overnight at room temperature, then was diluted with200 mL of ethyl ether and washed three times with water and once withsaturated brine. Drying over anhydrous magnesium sulfate and evaporationin vacuo afforded a residue which was chromatographed on silica gel,using first methylene chloride and then 1:1 methylene chloride/ethylacetate as eluents, to give 0.61 g (78.9%) of a colorless solid. NMR(DMSO-d₆) δ 8.35 (1H, m), 8.0 (3H, m), 7.4 (15H, m), 7.2 (2H, d), 6.9(2H, d), 5.05 (2H, s), 5.0 (2H, s), 4.6 (1H, m), 4.4-3.9 (6H, m), 3.7(2H, m), 3.1-2.6 (6H, m), 1.3 (9H, s), 1.2 (3H, t), 0.9 (6H, m); IR(KBr) ν 3300, 1690, 1640, 1510, 1240 cm⁻¹. Elemental analysis valueswere consistent with the assigned structure: ##STR123##

EXAMPLE 54 Preparation of3-[2-(N-tert-Butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1,4-dihydro-6-hydroxy-1-methylquinoline

To a mixture of 0.1 g of3-[2-(N-tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-6-hydroxy-1-methylquinoliniumiodide, 0.1 g of sodium bicarbonate, 15 mL of deaerated ethyl acetateand 10 mL of deaerated water was added, in one portion at roomtemperature under a nitrogen atmosphere, 0.15 g of sodium dithionite.The reaction mixture was stirred at that temperature for an additional 2hours. The organic layer was separated, washed with deaerated water andsaturated brine, dried over anhydrous sodium sulfate and evaporated invacuo to give 60 mg of a brown solid melting at 123°-125° C. NMR (CDCl₃)δ 6.8-6.4 (3H, m), 5.8 (1H, bs), 5.0 (1H, m), 4.1-4.0 (3H, m), 3.45 (2H,bs), 3.0 (3H, s), 2.4 (2H, m), 1.4 (9H, s), 1.25 (3H, t); IR (KBr) ν3400, 2970, 1730, 1685, 1510, 1370, 1230, 1155 cm⁻¹. Elemental analysisvalues were consistent with the assigned structure: ##STR124##

EXAMPLE 55 Preparation ofN-Benzoyl-3-(1,2-dihydro-1-methylquinolin-3-yl)alanine methyl ester

Repetition of the general procedure of EXAMPLE 21, substituting anequivalent quantity of3-(2-benzoylamino-2-methoxycarbonyl)ethyl-1-methylquinolinium iodide forthe3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide employed therein, affords a mixture of 1,2 and 1,4 dihydroderivatives. The title compound, which is the major product, has thestructural formula ##STR125##

EXAMPLE 56 Preparation ofN-Benzoyl-3-(1,4-dihydro-1-methylquinolin-3-yl(alanine methyl ester

When the general procedure of EXAMPLE 22 is repeated, substituting anequivalent quantity of3-(2-benzoylamino-2-methoxycarbonyl)ethyl-1-methylquinolinium iodide forthe3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide there employed, there is obtained, as the major product, thetitle compound of the formula ##STR126##

EXAMPLE 57 Preparation ofN-(tert-Butoxycarbonyl)-3-(3-carbamoyl-1,4-dihydropyridin-1-yl)-L-alanylglycineethyl ester

The procedure of EXAMPLE 25 is repeated, substituting an equivalentquantity ofL-1-[2-(tert-butoxycarbonyl)amino-2-(N-ethoxycarbonylmethyl)carbamoyl]ethyl-3-carbamoylpyridiniumchloride for theL-1-[5-(tert-butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoylpyridiniumchloride used therein. Obtained in this manner is the title compound ofthe formula ##STR127## The title compound can alternatively be named as1-[2-tert-butoxycarbonyl)amino-2-(N-ethoxycarbonylmethyl)carbamoyl]ethyl-3-carbamoyl-1,4-dihydropyridine.

EXAMPLE 58 Preparation ofN-(tert-Butoxycarbonyl)-3-(1,4-dihydro-6-methoxy-1-methylquinolin-3-yl)alanineethyl ester

The procedure of EXAMPLE 22 is followed, utilizing 0.1 g of3-[2-N-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-6-methoxy-1-methyl-3-quinoliniumiodide, 10 mL of water, 0.1 g of sodium bicarbonate, 15 mL of ethylacetate and 0.15 g of sodium dithionite. Prepared in this manner is thetitle compound of the formula: ##STR128##

EXAMPLE 59 Preparation ofN-(tert-Butoxycarbonyl)glycyl-3-(1,4-dihydro-1-methylquinolin-3-yl)alanineethyl ester

The procedure of EXAMPLE 22 was followed, utilizing 0.12 g (0.22 mmol)of3-{2-[N-(tert-butoxycarbonyl)glycyl]amino-2-ethoxycarbonyl}ethyl-1-methylquinoliniumiodide, 12 mL of water, 0.11 g (1.3 mmol) of sodium bicarbonate, 12 mLof methylene chloride and 0.16 g of sodium dithionite. There were thusobtained 72 mg of the title compound. NMR values were consistent withthe assigned structure: ##STR129##

EXAMPLE 60 Preparation of1-{5-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-5-methoxycarbonyl}pentyl-3-carbamoyl-1,4-dihydropyridine

When the general procedure of EXAMPLE 25 is repeated, using1-{5-[N-(tert-butoxycarbonyl)-L-tyrosyl]amino-5-methoxycarbonyl}pentyl-3-carbamoylpyridiniumchloride in place of theL-1-[5-(tert-butoxycarbonyl)amino-5-methoxycarbonyl]pentyl-3-carbamoylpyridiniumchloride, there is obtained the title compound of the formula:##STR130##

EXAMPLE 61 Preparation of1-{5-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-5-ethoxycarbonylmethylcarbamoyl}pentyl-3-carbamoyl-1,4-dihydropyridine

The general procedure of EXAMPLE 22 is repeated, using1-{5-[N-(tert-butoxycarbonyl)-L-tyrosyl]amino-5-ethoxycarbonylmethylcarbamoyl}pentyl-3-carbamoylpyridiniumchloride in place of the3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide employed in that Example. Obtained in this manner is the titlecompound of the formula: ##STR131##

EXAMPLE 62 Preparation ofL-[2-(tert-Butoxycarbonyl)amino-3-(3-carbamoyl-1-pyridinium)]propionyl-L-phenylalanyl-L-leucineethyl ester chloride

Following the general procedure of EXAMPLE 3, but substitutingN-(tert-butoxycarbonyl)-3-chloro-L-alanyl-L-phenylalanyl-L-leucine ethylester for the N-(tert-butoxycarbonyl)-3-chloro-L-alanylglycine ethylester there employed, affords the title compound of the formula:##STR132##

EXAMPLE 63 Preparation ofL-[2-(tert-Butoxycarbonyl)amino-3-(3-carbamoyl-1,4-dihydropyridin-1-yl)]propionyl-L-phenylalanyl-L-leucineethyl ester

The procedure of EXAMPLE 22 is repeated, usingL-[2-tert-butoxycarbonyl)amino-3-(3-carbamoyl-1-pyridinium)]propionyl-L-phenylalanyl-L-leucineethyl ester chloride in place of the3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide there employed. There is thus obtained the title compound of theformula: ##STR133##

EXAMPLE 64 Preparation of(S)-2-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycyl-L-phenylalanyl-L-leucineethyl ester chloride

A mixture of 1.0 g (1.02 mmol) ofO-benzyl-N-(tert-butoxycarbonyl)-L-tyrosyl-N.sup.ε-benzyloxycarbonyl-L-lysylglycyl-L-phenylalanyl-L-leucine ethyl ester,0.2 g of 10% palladium on carbon catalyst and 10 mL of dimethylformamidewas stirred vigorously under a hydrogen atmosphere for 2 hours. Thecatalyst was removed by filtration and to the filtrate was added 0.33 g(1.02 mmol) of 3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride. Thereaction mixture was stirred overnight at room temperature, thenevaporated. The residue was chromatographed on silica gel usingacetonitrile, 3% aqueous acetonitrile and 6% aqueous acetonitrilesuccessively as eluents, to give a brown oil. The oil was dissolved indistilled water, treated with active charcoal and lyophilized to give0.34 g (37.9%) of the title compound as a colorless solid. NMR (CD₃ OD)δ 9.45 (1H, bs), 9.1 (1H, d), 9.0 (1H, d), 8.2 (1H, dd), 7.3 (5H, bs),7.1 (2H, d), 6.7 (2H, d), 4.15 (2H, ql), 3.8 (2H, bs), 3.2-2.8 (4H, m),1.4 (9H, s), 1.25 (3H, t), 0.9 (6H, m). Elemental analysis wasconsistent with the assigned structure: ##STR134##

EXAMPLE 65 Preparation of(S)-2-[N-(tert-Butoxycarbonyl)-L-tyrosyl]amino-6-(3-carbamoyl-1,4-dihydropyridin-1-yl)hexanoylglycyl-L-phenylalanyl-L-leucineethyl ester

Repetition of the procedure of EXAMPLE 22, utilizing(S)-2-[N-(tert-butoxycarbonyl)-L-tyrosyl]amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycyl-L-phenylalanyl-L-leucineethyl ester chloride in place of the3-[2-(tert-butoxycarbonyl)amino-2-ethoxycarbonyl]ethyl-1-methylquinoliniumiodide there employed, affords the title compound of the structuralformula: ##STR135##

EXAMPLE 66 Preparation of(S)-1-[5-Amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoylpyridiniumchloride hydrochloride

A mixture of 0.1 mmol of(S)-1-[5-(tert-Butoxycarbonyl)amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoylpyridiniumchloride and 2 mL of ethanol saturated with HCl was stirred at 0° C. for20 minutes. The reaction mixture was diluted with 20 mL of dry ethylether, decanted and washed with ethyl ether. The resultant precipitatewas suspended in tert-butanol, evaporated and dried in vacuo to give thetitle compound as a white solid in 78.6% yield. NMR (DMSO-d₆) δ 9.85(1H, bs), 9.4 (2H, m), 9.0 (2H, dd), 8.5 (2H, m), 8.4-8.1 (3H, m), 4.8(2H, m), 4.2-3.8 (6H, m), 1.2 (3H, t). Elemental analysis was consistentwith the assigned structure: ##STR136## The product can also be named(S)-2-amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycine ethyl esterchloride hydrochloride.

EXAMPLE 67 Preparation of(S)-[2-Amino-6-(3-carbamoyl-1-pyridinium)]hexanoyl-L-phenylalanyl-L-leucineethyl ester chloride hydrochloride

The procedure of EXAMPLE 66 was repeated, using(S)-[2-(tert-butoxycarbonyl)amino-6-(3-carbamoyl-1-pyridinium)]hexanoyl-L-phenylalanyl-L-leucineethyl ester chloride as the starting material. The title compound wasobtained in 89.1% yield. NMR (DMSO-d₆) δ 9.8 (1H, bs), 9.35 (1H, bd),9.1-8.8 (2H, m), 8.7-8.1 (6H , m), 7.3 (5H, m), 4.7 (3H, m), 4.05 (2H,q), 3.8 (1H, m), 3.0 (2H, m), 1.2 (3H, t), 0.9 (6H, m). Elementalanalysis was consistent with the assigned structure: ##STR137##

EXAMPLE 68 Preparation of(S)-2-(L-Tyrosyl)amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycyl-L-phenylalanyl-L-leucineethyl ester chloride hydrochloride

The procedure of EXAMPLE 66 was repeated, using(S)-2-[N-(tert-butoxycarbonyl-L-tyrosyl]amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycyl-L-phenylalanyl-L-leucineethyl ester chloride as the starting material. The title compound wasobtained in 78.6% yield. NMR (DMSO-d₆) δ 9.75 (1H, bs), 9.3 (1H, d), 9.0(1H, d), 8.85 (1H, m), 8.6-8.0 (m), 7.25 (5H, m), 7.1 (2H, d), 6.7 (2H,d), 4.7 (2H, m), 1.15 (2H, t), 0.9 (6H, m). Elemental analysis wasconsitent with the assigned structure: ##STR138##

EXAMPLE 69 Preparation of(S)-1-[5-(L-Tyrosyl)amino-5-ethoxycarbonylmethylcarbamoyl]pentyl-3-carbamoylpyridiniumchloride hydrochloride:

Repetition of the procedure of EXAMPLE 66, but using(S)-1-{5-[N-(tert-butoxycarbonyl)-L-tyrosyl]amino-5-ethoxycarbonylmethylcarbamoyl}pentyl-3-carbamoylpyridiniumchloride as the starting material, affords the title compound of theformula: ##STR139## The product can also be named(S)-2-(L-tyrosyl)amino-6-(3-carbamoyl-1-pyridinium)hexanoylglycine ethylester chloride hydrochloride.

EXAMPLE 70 Preparation of 1,4-Dihydro Derivatives from the CorrespondingQuaternary Salts

The quaternary salts of the invention, e.g. the products of EXAMPLES 3,6, 9, 17, 20, 33, 39, 41, 42, 44, 46, 49, 51, 62, 64, 66, 67, 68 and 69may be selectively converted to the corresponding 1,4-dihydroderivatives using the following procedure:

A solution of 4 mmol of the selected quaternary salt and 4.6 mmol ofN-benzyl-1,2-dihydroisonicotinamide in anhydrous methanol is stirredunder nitrogen at 0° C. for 2 hours. The reaction mixture is evaporatedin vacuo at 30° C. and the residue is suspended in 1,2-dichloroethane,filtered and washed with the same solvent. The filtrate is evaporated invacuo to a small volume and then flash chromatographed over a column of150 mesh activated neutral aluminum oxide, eluting first with1,2-dichloroethane and then with 1:4 acetonitrile/1,2-dichloroethane.The solution is evaporated in vacuo at 30° C. and crystallized.

The peptides of structure (A) which are provided by this invention aretypically administered to mammals by incorporating the selected peptidein a pharmaceutical composition comprising the peptide or a non-toxicpharmaceutically acceptable salt thereof and a non-toxicpharmaceutically acceptable carrier therefor. The peptide or its salt isemployed in an effective amount, i.e. an amount sufficient to evoke thedesired pharmacological response. Thus, for example, the enkephalinanalogs of the invention may be employed in an analgesically effectiveamount; the LHRH agonist analogs of the invention may be used in anamount sufficient to control LH or FSH or to have the desired effect ofthe reproductive system (e.g. one or more of the physiological andparadoxical utilities disclosed in Nestor et al U.S. Pat. No.4,530,920); and so forth.

Suitable non-toxic pharmaceutically acceptable carriers for use with theselected peptide of structure (A) will be apparent to those skilled inthe art of pharmaceutical formulation. See, for example, Remington'sPharmaceutical Sciences, seventeenth edition, ed. Alfonso R. Gennaro,Mack Publishing Company, Easton, PA (1985). Obviously, the choice ofsuitable carriers will depend upon the exact nature of the particulardosage form selected, as well as upon the identity of the peptide to beadministered. The therapeutic dosage range can be estimated on the basisof animal tests, e.g. in the case of the LHRH agonist analogs, on thebasis of tests described in the Nestor et al patent referred tohereinabove. Naturally, such therapeutic dosage ranges will vary withthe particular peptide of structure (A) used, the size, species andcondition of the subject, the severity of the subject's condition, theparticular dosage form employed, the route of administration and thelike. And the quantity of given dosage form needed to deliver thedesired dose will of course depend upon the concentration of the peptideof structure (A) in any given pharmaceutical composition/dosage formthereof. In addition, the active ingredient may be formulated into asustained release carrier system and/or a route of administration may beselected to slowly release the chemical, e.g. subcutaneous implantationor transdermal delivery.

Routes of administration contemplated for the peptides of structure (A)and pharmaceutical compositions containing them are any of the routesgenerally used for treatment of the types of conditions for which thepeptides are administered. These include parenteral (intravenous,intramuscular, subcutaneous), vaginal, rectal, nasal, oral and buccalroutes. Appropriate dosage forms for these routes of administration willbe apparent to those skilled in the art; see, for example, Nestor et alU.S. Pat. No. 4,530,920. While any of these routes ofadministration/dosage forms are contemplated for peptides of structure(A) whose terminal amino and carboxyl functions are appropriatelyprotected by the protecting groups described herein for use in vivo,unprotected peptides having structure (A) may be administered inparental dosage forms suitable for direct injection into the brain.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims.

What is claimed is:
 1. A compound of the formula ##STR140## or anon-toxic pharmaceutically acceptable salt thereof, wherein Z is eithera direct bond or C₁ -C₆ alkylene and can be attached to the heterocyclicring via a ring carbon atom or via the ring nitrogen atom; R₁ is C₁ -C₇alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₂ aralkyl when Z is attached to a ringcarbon atom; R₁ is a direct bond when Z is attached to the ring nitrogenatom; R₂ and R₃, which can be the same or different, are selected fromthe group consisting of hydrogen, halo, cyano, C₁ -C₇ alkyl, C₁ -C₇alkoxy, C₂ -C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁ -C₇ haloalkyl, C₁-C₇ alkylthio, C₁ -C₇ alkylsulfinyl, C₁ -C₇ alkylsulfonyl, --CH═NOR'"wherein R'" is hydrogen or C₁ -C₇ alkyl, and --CONR'R" wherein R' andR", which can be the same or different, are each hydrogen or C₁ -C₇alkyl; R₄ is hydrogen or a carboxyl protective group and R₅ is hydrogenor an amino protective group, said carboxyl protective group and saidamino protective group being designed to protect the carboxyl and aminofunctions during synthesis or to improve lipoidal characteristics andprevent premature metabolism of said functions in vivo; and the dottedlines indicate that the compound of formula (I) contains a 1,4- or1,6-dihydropyridine ring system.
 2. A compound according to claim 1wherein Z is --CH₂ --.
 3. A compound according to claim 1 wherein Z is--(CH₂)₄ --.
 4. A compound according to claim 1 wherein R₁ is a directbond and Z is attached to the ring nitrogen atom.
 5. A compoundaccording to claim 4 wherein one of R₂ and R₃ is hydrogen.
 6. A compoundaccording to claim 5 wherein the other of R₂ and R₃ is --CONH₂.
 7. Acompound according to claim 1 wherein R₁ is methyl.
 8. A compoundaccording to claim 1 wherein Z is attached to a ring carbon atom.
 9. Acompound according to claim 8 wherein R₁ is methyl.
 10. A compoundaccording to claim 8 wherein at least one of R₂ and R₃ is hydrogen. 11.A compound according to claim 10 wherein both R₂ and R₃ are hydrogen.12. A compound according to claim 1 wherein R₄ is a carboxyl protectivegroup.
 13. A compound according to claim 1 wherein R₅ is an aminoprotective group.
 14. A compound according to claim 1 containing a1,4-dihydropyridine ring system.
 15. A compound according to claim 1containing a 1,6-dihydropyridine ring system.
 16. A compound accordingto claim 1 wherein ##STR141##
 17. A compound according to claim 1wherein ##STR142##
 18. A compound according to claim 1 wherein##STR143##
 19. A compound according to claim 1 wherein ##STR144##
 20. Acompound according to claim 1 having the formula ##STR145##